Angiotensin peptide-carrier conjugates and uses thereof

ABSTRACT

The present invention provides conjugates of peptide derivatives of the mammalian peptide hormones angiotensinogen, angiotensin I and angiotensin II, presented in a repetitive scaffold by coupling the peptide derivatives to a carrier, particularly a virus-like particle (VLP). The invention also provides methods of producing such conjugates, and immunotherapeutic uses of the resulting immunogen conjugates for the therapy and prophylaxis of conditions associated with the renin-activated angiotensin system.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication Nos. 60/326,998, filed Oct. 5, 2001; 60/331,045, filed Nov.7, 2001; and 60/396,637, filed Jul. 19, 2002. The present applicationalso claims priority under 35 U.S.C. § 120 to U.S. application Ser. No.10/050,902, filed Jan. 18, 2002, and to International Application No.PCT/IB02/00166, filed Jan. 21, 2002, which designated the United Statesof America and which was published in English under PCT Article 21(2) asWO 02/056905 on Jul. 25, 2002. The disclosures of all of theabove-referenced applications are incorporated by reference herein intheir entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is in the fields of medicine, public health,immunology, molecular biology and virology.

2. Related Art

The arterial blood pressure of mammals is mostly controlled by abiochemical cascade known as the renin-angiotensin-System (RAS). It isinitiated by the release of renin from the epitheloid cells of thejuxtaglomerular apparatus of the kidney following a fall in arterialblood pressure. Renin enzymatically cleaves the peptide angiotensinogen(amino acid sequence:Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn) which issecreted into the serum by the liver. This cleavage leads to theformation of the decapeptide angiotensin I (amino acid sequence:Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu). The angiotensin convertingenzyme (ACE) which is present in the endothelium of the lungs cleaveswithin seconds the two C-terminal amino acids of ATI to give rise toangiotensin II (amino acid sequence: Asp-Arg-Val-Tyr-Ile-His-Pro-Phe).Whereas angiotensin I is very short lived within the body and has no oronly mild vasoconstrictor activity, angiotensin II has a profound effecton the circulatory system as well as on the endocrine system. Elevatedlevels of RAS-activated angiotensin II cause vasoconstriction, renalretention of salt and water, both of which contribute to increasedarterial pressure (hypertension) which can lead to cardiovasculardamage. Possible clinical manifestations of hypertension are stroke,infarction, congestive heart failure, kidney failure or retinalhemorrhage.

According to the U.S. Centers for Disease Control and Prevention (CDC),congestive heart failure is a major chronic disease for older adults,accounting for about 260,000 deaths a year in the US. In 1995, $3.4billion was paid by Medicare for heart failure. Although drugs areavailable for the treatment of hypertension, control of hypertension isonly obtained in around half of the treated hypertensive patients. Thisis partially due to non-compliance of the patient or ineffectiveness ofthe used drugs.

Current treatment of hypertension includes intervention of the RASsystem using small organic molecules. Main targets are renin, ACE andthe receptors for angiotensin II. ACE inhibitors include lisinopril®,captopril® and enalapril®, however, these drugs have not been entirelysuccessful. Firstly they do not seem to entirely block ACE activity andsecondly the generation by ACE of other biologically active peptides,including bradykinin, is also affected, which is undesirable. Thesedrugs can induce side effects such as dry cough and a first dosehypotensive effect with dizziness and possible fainting. Angiotensin IIreceptor antagonists include losartan®, valsartan® and isbesaftan® whichact specifically on the AT1 angiotensin receptor; they therefore blockthe dominant vasoconstrictor effects of angiotensin II, and are bettertolerated but do not affect other actions of the angiotensin hormones.However, angiotensin II receptor antagonists as well as ACE inhibitorsneed to be taken on a regular basis, often for long periods, such as forthe majority of adult life which at least partially explains poorpatient compliance. Therefore, there is a clear need for therapies ofhypertension which are effective, well tolerated and connected with ahigh compliance of the patient.

A potential approach in treating or preventing diseases or disordersassociated with the activity of a hormone is to neutralize the effectsof the hormone within the patient by immunotherapy, i.e., by immunizingthe patient against the hormone or enzymes which are involved in thegeneration of the hormone such that the activity of the hormone isneutralized or its levels are reduced by specific anti-hormone oranti-enzyme antibodies. Such antibodies may be exogenously administeredin passive immunization or they may be generated in situ by activeimmunization using an immunogen based on the hormone or the relatingenzyme.

The feasibility of vaccination against components of the RAS to modulatehypertension has been shown in animal models (for a review, see Michel,Am. Heart J. 117:756 (1989)). Vaccination against renin was effective inreducing blood pressure, however the animals suffered from autoimmunenephritis. (Michel et al., Circulation 81:1899 (1990); Lo et al.,Hypertension 16:80 (1990)). Data on active immunization againsthomologous ACE is very limited. One report describes the vaccination ofrabbits but only 1 out of 50 animals made detectable anti-ACE antibodies(Soffer, Fed. Proc. 42:2735 (1983)). Passive transfer of immune serumagainst ACE can decrease blood pressure in rabbits but leads to animmunoallergic response with pulmonary edema, possibly because ACE isexpressed in a membrane-bound form in the lung (Cadwell, FEBS Lett.63:82 (1976)). No reports are available on active immunization againstangiotensinogen, however several studies explored the feasibility ofvaccination against angiotensin I and angiotensin II. Two studiesreported a blood pressure effect (Christlieb, J. Clin. Invest. 48:1506(1969); Gardiner, Br. J. Pharmacol. 129:1178 (2000)) in vaccinatedanimals and no autoimmunity was noted. However the majority ofvaccination studies with angiotensin peptides were negative, possiblybecause the induced titers against angiotensin peptides were too low orbecause the specificity of the induced antibodies was not optimal. It islikely that a vaccine which only targets angiotensin II does not havethe same effect on the RAS as a vaccine which induces antibodies againstangiotensin II as well as angiotensin I and possibly also the precursorangiotensinogen.

WO 98/58952 describes the treatment with a conjugate containing anangiotensin I conjugated to tetanus toxoid, which leads to the inductionof angiotensin-specific antibodies in rats if applied in conjunctionwith an adjuvant such as aluminium hydroxide. Adjuvants are often toxicor at least irritating. The only adjuvants allowed for human use to dateare mineral salts (aluminum hydroxide, aluminium phosphate, calciumphosphate) and virosomes. The adjuvant most frequently used in humans isaluminum hydroxide (Alum). Although it is considered as safe, it remainsin the body for an extended period of time forming a depot. Consequencesof such depot-formation are still poorly understood, therefore attemptsshould be made to avoid Alum in future vaccines without loosing theirimmunogenicity.

Therefore, there remains a need in the art to provide conjugates leadingto the induction of high antibody titers even in the absence ofadjuvants.

SUMMARY OF THE INVENTION

We have now developed potent immunogens for the induction of antibodiesspecific for angiotensinogen, angiotensin I or angiotensin II (referredto herein collectively as “angiotensin peptides”), which are effectiveeven without the use of adjuvants and which allow the development ofantibodies in vivo that specifically target one or more angiotensinpeptides, such as angotensinogen, angiotensin I or angiotensin II. Theimmunogens consist of angiotensin peptide moieties which are bound tovirus-like particles (VLP). This results in a highly immunogenicrepetitive antigen array which is able to stimulate antibody formationeven without the use of adjuvants. Depending on the amino acid sequenceof the angiotensin peptide moieties used, high antibody titers areinduced, and, moreover, can be specifically induced against the N- orC-terminal ends of angiotensinogen, angiotensin I or angiotensin II.This allows the specific targeting of only one species of angiotensinpeptides or a combination thereof. The immunogens of the presentinvention thus can be used in an immunotherapeutic approach to combatconditions associated with elevated levels of angiotensin II produced bythe RAS.

Without intending to be limited to any particular theory of operation ormechanism, the conjugates and conjugates of the invention can induceantibodies which bind to more than one angiotensin peptide species,thereby blocking all relevant species of angiotensin at the same time.Alternatively, the induced antibodies could specifically to theC-terminus of angiotensinogen, angiotensin I or angiotensin II. Underthese conditions, the induced antibodies will block activation ofangiotensinogen or angiotensin I by renin or ACE, respectively.Nevertheless, proteases different from ACE or renin, such asendopeptidases and aminopeptidases, can degrade angiotensinogen,angiotensin I or angiotensin II from the N-terminus thus preventing theaccumulation of antibody-bound intact angiotensinogen, angiotensin I orangiotensin II.

Thus, by the invention, immunogens are provided that comprise one ormore angiotensin peptides or peptide moieties, or derivatives thereof,bound to one or more core particles, preferably one or more virus-likeparticles (VLPs), to form conjugates having the structure of ordered andrepetitive arrays. Core particles containing a first attachment site,and angiotensin peptides or derivatives thereof containing a secondattachment site, are associated through said first and second attachmentsites to form such ordered and repetitive arrays. The interactionbetween the first and second sites may be direct, or may involve atleast one other molecule, e.g., a linker.

In one embodiment, the first attachment site naturally occurs in thecore particle. Alternatively, the first attachment site is added bychemical coupling or by recombinant techniques. Preferred firstattachment sites comprise amino groups, carboxyl groups or sulfhydrylgroups. Preferred amino acids comprising a first attachment site areselected from lysine, arginine, cysteine, aspartate, glutamate tyrosineand histidine. Particularly preferred are lysine residues.

Suitable second attachment sites on the angiotensin peptides orderivatives thereof are amine, amide, carboxyl and sulfhydryl groups.There is a wide range of compounds that have been developed to enablecrosslinking of peptides/proteins or conjugation of protein toderivatized molecules, by forming a covalent bond with a reactive groupof a protein molecule of the core particle.

Core particles with a first attachment site of the invention include anyparticle suitable for the formation of ordered repetitive arrays. Insome embodiments such core particles include virus-like particles(VLPs), bacteriophage, bacteriophage virus like particles, pili, and thelike. In certain embodiments these are HbcAg VLPs, bacteriophage VLP andtype I pili. The invention also provides variant forms of the coreparticles that remain able to form ordered repetitive structure. Variantforms include recombinant and natural forms, and mutant forms of coreparticles. In certain embodiments, the mutant forms of the core particleinclude those where the type of first attachment site, or number of saidsites, differ from the parent. Alteration of the number of lysineresidues on the core particle are particularly preferred.

In certain embodiments, conjugates of the invention comprise angiotensinpeptide moieties which are chemically coupled to virus-like particles(VLP). This results in a highly immunogenic repetitive antigen arraywhich is able to stimulate antibody formation even without the use ofadjuvants. Depending on the amino acid sequence of the angiotensinpeptide moieties used, high antibody titers are induced, and, moreover,can be specifically induced against the N- or C-terminal ends ofangiotensinogen, angiotensin I or angiotensin II. This allows tospecifically target only one species of angiotensin peptides or acombination thereof. The inventive immunogens can be used in animmunotherapeutic approach to combat conditions associated with elevatedlevels of angiotensin II produced by the RAS.

The present invention thus provides conjugates comprising a coreparticle and one or more angiotensin peptides or angiotensin peptidemoieties, suitable for use in inducing immune responses. The inventionalso provides conjugates comprising such conjugates of the invention andone or more additional components such as one or more excipients orcarriers, suitably one or more pharmaceutically acceptable excipients orcarriers. Conjugates and conjugates of the invention include vaccineconjugates or conjugates, with or without additional pharmaceuticallyacceptible excipients or adjuvants. For example, the present inventionalso provides vaccine conjugates comprising an immunologically effectiveamount of the one or more of the conjugates or conjugates of the presentinvention together with a pharmaceutically acceptable diluent, carrieror excipient. In a further embodiment, the vaccine further comprises atleast one adjuvant, such as Alum or incomplete Freund's adjuvant. Theinvention also provides methods of immunizing and/or treating an animal,preferably a mammal such as a human, comprising administering to theanimal an immunologically effective amount of conjugates, conjugates, orvaccines of the invention thereby inducing an immune response againstthe conjugates or conjugates. Animals may be suitably immunized with theconjugates or conjugates of the invention by any art-known route ofadministration, including but not limited to subcutaneously,intramuscularly, intranasally, intradermally, intravenously,transdermally, transmucosally, orally, or directly into a lymph node.Intranasal immunization is a particularly suitable route; this type ofadministration leads not only to high antibody titers encompassing IgAas indicated in the examples but also, by avoiding painful immunizationprocedures (e.g intramuscular) is more acceptible to the patient andleads to improved compliance.

Conjugates and conjugates of the invention induce immune responses,including the production of antibodies. Therefore, in anotherembodiment, the invention provides methods of producing antibodiesagainst one or more angiotensin peptides or angiotensin peptidemoieties. Such antibodies of the invention are useful in treatment orprevention of physical disorders associated with the RAS, and for thedetection of angiotensin peptides or angiotensin peptide moieties, forexample in the methods of diagnosing physical disorders associated withthe presence of one or more components of the RAS in the tissues orcirculation of an animal.

In a related embodiment, the invention is useful for the prevention ortreatment of diseases, disorders or conditions associated with the RAS,including but not limited to stroke, infarction, congestive heartfailure, kidney failure, retinal hemorrhage and the like. Immunizationwith the conjugates or conjugates of the invention results in an immuneresponse against the one or more angiotensin peptides or angiotensinpeptide moieties, such that immune molecules, particularly antibodies,bind the angiotensin peptides or angiotensin peptide moieties. Passivetransfer of antibodies is also useful for the treatment and preventionof disorders associated with the RAS.

We have found that conjugates of angiotensin peptides or angiotensinpeptide moieties attached to virus-like particles (VLPs) induce highangiotensin-specific IgG antibodies. The present invention thereforeprovides a therapeutic for physical disorders associated with the RAS,which is based, in a very preferred embodiment, on a ordered andrepetitive VLP-angiotensin peptide/moiety conjugate. This therapeutic isable to induce high titers of anti-angiotensin antibodies in avaccinated animal. High antibody titers are induced even in the absenceof adjuvants and encompass not only IgG but also IgA subtypes.Furthermore, this therapeutic surprisingly is not associated withinduction of potentially pathologic immune responses such asinflammation. Therapeutic conjugates of the invention comprise at leastone angiotensin peptide or angiotensin peptide moiety and a VLP,preferably a VLP of a RNA-phage, or at least angiotensin peptide orangiotensin peptide moiety and an alternative core particle such asHbcAg or pili.

Other embodiments of the present invention will be apparent to one ofordinary skill in light of what is known in the art, the followingdrawings and description of the invention, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 is an ELISA analysis of IgG antibodies specific for the Angio 1peptide and angotensin II in sera of mice immunized with Angio 1, Angio2, Angio 3 or Angio 4 peptides coupled to Qβ capsid protein.

FIG. 2 is an ELISA analysis of IgG antibodies specific for the Angio 2peptide and angotensin I in sera of mice immunized with Angio 1, Angio2, Angio 3 or Angio 4 peptides coupled to Qβ capsid protein.

FIG. 3 is an ELISA analysis of IgG antibodies specific for the Angio 1peptide and angotensin II in sera of mice immunized with Angio 5, Angio6, Angio 7, Angio 8 or Angio 9 peptides coupled to Qβ capsid protein.

FIG. 4 is an ELISA analysis of IgG antibodies specific for the Angio 2peptide and angotensin I in sera of mice immunized with Angio 5, Angio6, Angio 7, Angio 8 or Angio 9 peptides coupled to Qβ capsid protein.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

In the description that follows, a number of terms used in the field ofmolecular biology, immunology and medicine are extensively utilized. Inorder to provide a clearer and consistent understanding of thespecification and claims, including the scope to be given such terms,the following non-limiting definitions are provided.

Active immunization: As used herein, the term “active immunization”refers to the induction of an immune response in an individual,typically an animal, elicited by the administration of an immunogen,vaccine, antigen or angiotensin peptide-carrier conjugate. By contrast,passive immunization refers to the conferral of immunity in anindividual by the transfer of immune molecules or cells into saidindividual.

Alphavirus: As used herein, the term “alphavirus” refers to any of theRNA viruses included within the genus Alphavirus. Descriptions of themembers of this genus are contained in Strauss and Strauss, Microbiol.Rev., 58:491-562 (1994). Examples of alphaviruses include Aura virus,Bebaru virus, Cabassou virus, Chikungunya virus, Easter equineencephalomyelitis virus, Fort morgan virus, Getah virus, Kyzylagachvirus, Mayoaro virus, Middleburg virus, Mucambo virus, Ndumu virus,Pixuna virus, Tonate virus, Triniti virus, Una virus, Western equineencephalomyelitis virus, Whataroa virus, Sindbis virus (SIN), Semlikiforest virus (SFV), Venezuelan equine encephalomyelitis virus (VEE), andRoss River virus.

Amino acid linker: An “amino acid linker,” or also just termed “linker”within this specification, as used herein, either associates the antigenor antigenic determinant with the second attachment site, or morepreferably, already comprises or contains the second attachment site,typically—but not necessarily—as one amino acid residue, preferably as acysteine residue. The term “amino acid linker” as used herein, however,does not intend to imply that such an amino acid linker consistsexclusively of amino acid residues, even if an amino acid linkerconsisting of amino acid residues is a preferred embodiment of thepresent invention. The amino acid residues of the amino acid linker are,preferably, composed of naturally occuring amino acids or unnaturalamino acids known in the art, all-L or all-D or mixtures thereof.However, an amino acid linker comprising a molecule with a sulfhydrylgroup or cysteine residue is also encompassed within the invention. Sucha molecule comprise preferably a C1-C6 alkyl-, cycloalkyl (C5,C6), arylor heteroaryl moiety. However, in addition to an amino acid linker, alinker comprising preferably a C1-C6 alkyl-, cycloalkyl (C5,C6), aryl-or heteroaryl-moiety and devoid of any amino acid(s) shall also beencompassed within the scope of the invention. Association between theantigen or antigenic determinant or optionally the second attachmentsite and the amino acid linker is preferably by way of at least onecovalent bond, more preferably by way of at least one peptide bond.

Angiotensin Peptide Moiety: As used herein, the term “angiotensinpeptide moiety” refers to any moiety, whether or not the moiety has thebiological activity of a native angiotensin in vivo (e.g., nativehormone activity at the receptors, including both angiotensins I andII), which is capable of acting as an immunomimic of native angiotensinpeptides (i.e., which immunologically mimics angiotensin so as togenerate antibodies which bind to native angiotensin peptides). Thus,such a moiety may conveniently comprise an angiotensin peptide,preferably angiotensinogen, angiotensin I (a decapeptide of formulaAsp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) or angiotensin II (anoctapeptide of formula Asp-Arg-Val-Tyr-Ile-His-Pro-Phe), or afunctionally equivalent variant thereof. Hence, “angiotensin peptidemoiety” encompasses “angiotensin peptide” as that term is definedherein. Such functionally equivalent variants may include modificationsof the angiotensin I or II sequence by single or multiple amino acidsubstitution, addition or deletion and also sequences where the aminoacid residues are chemically modified, but which nonetheless retainangiotensin immunogenic activity. Such functionally (or immunologically)equivalent variants may occur as natural biological variations, or theymay be prepared using known and standard techniques for example bychemical synthesis or modification, mutagenesis, e.g., site-directed orrandom mutagenesis, etc. For purposes of this definition, a key featureas regards the modification is that the angiotensin peptide retains theability to act as immunomimic of native angiotensin. Thus for example,an amino acid may be replaced by another which preserves thephysicochemical character of the angiotensin peptide or its epitope(s),e.g. in terms of charge density, hydrophilicity/hydrophobicity, size andconfiguration and hence preserve the immunological structure. “Addition”variants may include N- or C-terminal fusions as well as intrasequenceinsertion of single or multiple amino acids. Deletions may beintrasequence or may be truncations from the N- or C-termini. Preferreddeletion mutants are those that allow induction of N- or preferablyC-terminal antibodies. Such antibodies may prevent generation of activeangiotensin II but still allow for degradation of antibody-boundangiotensinogen, angiotensin I or angiotensin II.

Angiotensin Peptide: As used herein, the term “angiotensin peptide”includes all, preferably native, angiotensin peptides and theirfunctionally equivalent variants. Hence, “angiotensin peptide” can beconsidered a subset of “angiotensin peptide moiety” as defined herein.As a practical matter, whether a given variant of an angiotensin peptide(or angiotensin peptide moiety) is “functionally equivalent” to a,preferably native, angiotensin peptide may be determined by a variety ofassay methods for determining the biological activity of an angiotensinpeptide. Certain of these assay methods are described herein, and otherswill be readily familiar to one of ordinary skill in the art.

Antibody: As used herein, the term “antibody” refers to molecules whichare capable of binding an epitope or antigenic determinant. The term ismeant to include whole antibodies and antigen-binding fragments thereof,including single-chain antibodies. Such antibodies include human antigenbinding antibody fragments and include, but are not limited to, Fab,Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv) and fragments comprising either a V_(L) orV_(H) domain. The antibodies can be from any animal origin includingbirds and mammals. Preferably, the antibodies are mammalian e.g. human,murine, rabbit, goat, guinea pig, camel, horse and the like, or othersuitable animals e.g. chicken. As used herein, “human” antibodiesinclude antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or from animals transgenic for one or more humanimmunoglobulins and that do not express endogenous immunoglobulins, asdescribed, for example, in U.S. Pat. No. 5,939,598, the disclosure ofwhich is incorporated herein by reference in its entirety.

Antigen: As used herein, the term “antigen” refers to a molecule capableof being bound by an antibody or a T cell receptor (TCR) if presented byMHC molecules. The term “antigen”, as used herein, also encompassesT-cell epitopes. A T-cell epitope is recognized by a T-cell receptor inthe context of a MHC class I, present on all cells of the body excepterythrocytes, or class II, present on immune cells and in particularantigen presenting cells. This recognition event leads to activation ofT-cells and subsequent effector mechanisms such as proliferation of theT-cells, cytokine secretion, perforin secretion etc. An antigen isadditionally capable of being recognized by the immune system and/orbeing capable of inducing a humoral immune response and/or cellularimmune response leading to the activation of B- and/or T-lymphocytes.This may, however, require that, at least in certain cases, the antigencontains or is linked to a T_(H) cell epitope and is given in adjuvant.An antigen can have one or more epitopes (B- and T-epitopes). Thespecific reaction referred to above is meant to indicate that theantigen will preferably react, typically in a highly selective manner,with its corresponding antibody or TCR and not with the multitude ofother antibodies or TCRs which may be evoked by other antigens. Antigensas used herein may also be mixtures of several individual antigens.

Antigenic determinant: As used herein, the term “antigenic determinant”is meant to refer to that portion of an antigen that is specificallyrecognized by either B- or T-lymphocytes. B-lymphocytes respond toforeign antigenic determinants via antibody production, whereasT-lymphocytes are the mediator of cellular immunity. Thus, antigenicdeterminants or epitopes are those parts of an antigen that arerecognized by antibodies, or in the context of an MHC, by T-cellreceptors. An antigenic determinant contains one or more epitopes.Allergens also serve as antigens in vertebrate animals.

Association: As used herein, the term “association” as it applies to thefirst and second attachment sites, refers to the binding of the firstand second attachment sites that is preferably by way of at least onenon-peptide bond. The nature of the association may be covalent, ionic,hydrophobic, polar or any combination thereof, preferably the nature ofthe association is covalent.

Attachment Site, First: As used herein, the phrase “first attachmentsite” refers to an element of the core particle being of non-natural ornatural origin, to which the second attachment site located on theantigen or antigenic determinant may associate. The first attachmentsite may be a protein, a polypeptide, an amino acid, a peptide, a sugar,a polynucleotide, a natural or synthetic polymer, a secondary metaboliteor compound (biotin, fluorescein, retinol, digoxigenin, metal ions,phenylmethylsulfonylfluoride), or a combination thereof, or a chemicallyreactive group thereof. The first attachment site is located, typicallyand preferably on the surface, of the core particle such as, preferablythe virus-like particle. Multiple first attachment sites are present onthe surface of the core and virus-like particle, respectively, typicallyin a repetitive configuration.

Attachment Site, Second: As used herein, the phrase “second attachmentsite” refers to an element associated with the antigen or antigenicdeterminant to which the first attachment site located on the surface ofthe core particle and virus-like particle, respectively, may associate.The second attachment site of the antigen or antigenic determinant maybe a protein, a polypeptide, a peptide, a sugar, a polynucleotide, anatural or synthetic polymer, a secondary metabolite or compound(biotin, fluorescein, retinol, digoxigenin, metal ions,phenylmethylsulfonylfluoride), or a combination thereof, or a chemicallyreactive group thereof. At least one second attachment site is presenton the antigen or antigenic determinant. The term “antigen or antigenicdeterminant with at least one second attachment site” refers, therefore,to an antigen or antigenic construct comprising at least the antigen orantigenic determinant and the second attachment site. However, inparticular for a second attachment site, which is of non-natural origin,i.e. not naturally occurring within the antigen or antigenicdeterminant, these antigen or antigenic constructs comprise an “aminoacid linker”.

Bound: As used herein, the term “bound” refers to binding or attachmentthat may be covalent, e.g., by chemically coupling, or non-covalent,e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc.Covalent bonds can be, for example, ester, ether, phosphoester, amide,peptide, imide, carbon-sulfur bonds, carbon-phosphorus bonds, and thelike. The term “bound” is broader than and includes terms such as“coupled,” “fused” and “attached.”

Coat protein(s): As used herein, the term “coat protein(s)” refers tothe protein(s) of a bacteriophage or a RNA-phage capable of beingincorporated within the capsid assembly of the bacteriophage or theRNA-phage. However, when referring to the specific gene product of thecoat protein gene of RNA-phages the term “CP” is used. For example, thespecific gene product of the coat protein gene of RNA-phage Qβ isreferred to as “Qβ CP”, whereas the “coat proteins” of bacteriophageQβcomprise the “Qβ CP” as well as the A1 protein. The capsid ofBacteriophage Qβ is composed mainly of the Qβ CP, with a minor contentof the A1 protein. Likewise, the VLP Qβ coat protein contains mainly QβCP, with a minor content of A1 protein.

Core particle: As used herein, the term “core particle” refers to arigid structure with an inherent repetitive organization. A coreparticle as used herein may be the product of a synthetic process or theproduct of a biological process.

Effective Amount: As used herein, the term “effective amount” refers toan amount necessary or sufficient to realize a desired biologic effect.An effective amount of the composition would be the amount that achievesthis selected result, and such an amount could be determined as a matterof routine by a person skilled in the art. For example, an effectiveamount for treating an immune system deficiency could be that amountnecessary to cause activation of the immune system, resulting in thedevelopment of an antigen specific immune response upon exposure toantigen. The term is also synonymous with “sufficient amount.”

The effective amount for any particular application can vary dependingon such factors as the disease or condition being treated, theparticular composition being administered, the size of the subject,and/or the severity of the disease or condition. One of ordinary skillin the art can empirically determine the effective amount of aparticular composition of the present invention without necessitatingundue experimentation.

Epitope: As used herein, the term “epitope” refers to basic element orsmallest unit of recognition by an individual antibody or T-cellreceptor, and thus the particular domain, region or molecular structureto which said antibody or T-cell receptor binds. An antigen may consistof numerous epitopes while a hapten, typically, may possess fewepitopes.

Fusion: As used herein, the term “fusion” refers to the combination ofamino acid sequences of different origin in one polypeptide chain byin-frame combination of their coding nucleotide sequences. The term“fusion” explicitly encompasses internal fusions, i.e., insertion ofsequences of different origin within a polypeptide chain, in addition tofusion to one of its termini.

Heterologous sequence: As used herein, the term “heterologous sequence”refers to a second sequence of nucleic acid or protein that is notnormally found with said nucleic acid or protein and is, usually,artificially added to the sequence in order to confer particularproperties. In one example, heterologous amino acids may be added torecombinant capsid proteins for the purposes of purification of theprotein, or to serve as a first attachment site.

Immune response: As used herein, the term “immune response” refers toany action by the immune system of an individual that is directedagainst a molecule or compound, such as an antigen. In mammals, theimmune response includes both the activities of cells and the productionof soluble molecules such as cytokines and antibodies. The term thusincludes a humoral immune response and/or cellular immune responseleading to the activation or proliferation of B- and/or T-lymphocytes.In some instances, however, the immune responses may be of low intensityand become detectable only when using at least one substance inaccordance with the invention. “Immunogenic” refers to an agent used tostimulate the immune system of a living organism, so that one or morefunctions of the immune system are increased and directed towards theimmunogenic agent. An “immunogenic polypeptide” is a polypeptide thatelicits a cellular and/or humoral immune response, whether alone orlinked to a carrier in the presence or absence of an adjuvant.

Immune Deviation: As used herein, the term immune deviation refers tothe stimulation of an immune response that is of a different nature to apreexisting immune response. For example, an individual possessing aT_(H)2 immune response against an allergen such that IgE antibodies areproduced upon exposure to the allergen may be induced, by embodiments ofthe present invention, to produce a T_(H)1 immune response against theallergen. Such T_(H)1 response will counteract the allergy inducingT_(H)2 response and so alleviate allergic disease.

Immunotherapeutic: As used herein, the term “immunotherapeutic” refersto a conjugate for the treatment of diseases, disorders or conditions.More specifically; the term is used to refer to a method of treatmentwherein a beneficial immune response is generated by vaccination.

Immunologically effective amount: As used herein, the term“Immunologically effective amount” refers to an amount of a conjugatesufficient to induce an immune response in an individual when introducedinto that individual. The amount of a conjugate necessary to beimmunologically effective varies according many factors including to theconjugate, the presence of other components in the conjugate (e.g.adjuvants), the antigen, the route of immunization, the individual, theprior immune or physiologic state etc.

Individual: As used herein, the term “individual” refers tomulticellular organisms and includes both plants and animals. Preferredmulticellular organisms are animals, more preferred are vertebrates,even more preferred are mammals, and most preferred are humans.

Isolated: As used herein, when the term “isolated” is used in referenceto a molecule, the term means that the molecule has been removed fromits native environment. For example, a polynucleotide or a polypeptidenaturally present in a living animal is not “isolated,” but the samepolynucleotide or polypeptide separated from the coexisting materials ofits natural state is “isolated.” Further, recombinant DNA moleculescontained in a vector are considered isolated for the purposes of thepresent invention. Isolated RNA molecules include in vivo or in vitroRNA replication products of DNA and RNA molecules. Isolated nucleic acidmolecules further include synthetically produced molecules.Additionally, vector molecules contained in recombinant host cells arealso isolated. Thus, not all “isolated” molecules need be “purified.”

Immunotherapeutic: As used herein, the term “immunotherapeutic” is aconjugate that comprises immune molecules and/or elicits an immuneresponse for the treatment of diseases or disorders.

Individual: As used herein, the term “individual” refers tomulticellular organisms and includes both plants and animals. Preferredmulticellular organisms are animals, more preferred are vertebrates,even more preferred are mammals, and most preferred are humans.

Low or undetectable: As used herein, the phrase “low or undetectable,”when used in reference to gene expression level, refers to a level ofexpression which is either significantly lower than that seen when thegene is maximally induced (e.g., at least five fold lower) or is notreadily detectable by the methods used in the following examplessection.

Lectin: As used herein, proteins obtained particularly from the seeds ofleguminous plants, but also from many other plant and animal sources,that have binding sites for specific mono- or oligosaccharides. Examplesinclude concanavalin A and wheat-germ agglutinin, which are widely usedas analytical and preparative agents in the study of glycoprotein.

Mimotope: As used herein, the term “mimotope” refers to a substancewhich induces an immune response to an antigen or antigenic determinant.Generally, the term mimotope will be used with reference to a particularantigen. For example, a peptide which elicits the production ofantibodies to a phospholipase A₂ (PLA₂) is a mimotope of the antigenicdeterminant to which the antibodies bind. A mimotope may or may not havesubstantial structural similarity to or share structural properties withan antigen or antigenic determinant to which it induces an immuneresponse. Methods for generating and identifying mimotopes which induceimmune responses to particular antigens or antigenic determinants areknown in the art and are described elsewhere herein.

Mutein: As used herein, the term “mutein” refers to a protein orpolypeptide differing by one or more amino acids from a given reference(e.g. natural, wild type, etc.) polypeptide

Natural origin: As used herein, the term “natural origin” means that thewhole or parts thereof are not synthetic and exist or are produced innature. Preferably, as used herein, the term “natural origin” means thatthe whole is not synthetic and exist or is produced in nature.

Non-natural: As used herein, the term generally means not from nature,more specifically, the term means from the hand of man.

Non-natural molecular scaffold: As used herein, the phrase “non-naturalmolecular scaffold” refers to any product made by the hand of man thatserves to provide a rigid and repetitive array of first attachmentsites. Ideally but not necessarily, these first attachment sites are ina geometric order. The non-natural molecular scaffold may be organic ornon-organic and may be synthesized chemically or through a biologicalprocess, in part or in whole. The non-natural molecular scaffold iscomprised of: (a) a core particle, either of natural or non-naturalorigin; and (b) at least one first attachment site. Non-natural origin:As used herein, the term “non-natural origin” generally means syntheticor not from nature; more specifically, the term means from the hand ofman.

Ordered and repetitive antigen or antigenic determinant array: As usedherein, the term “ordered and repetitive antigen or antigenicdeterminant array” generally refers to a repeating pattern of antigen orantigenic determinant, characterized by a typically and preferablyuniform spacial arrangement of the antigens or antigenic determinantswith respect to the core particle and virus-like particle, respectively.In one embodiment of the invention, the repeating pattern may be ageometric pattern. Typical and preferred examples of suitable orderedand repetitive antigen or antigenic determinant arrays are those whichpossess strictly repetitive paracrystalline orders of antigens orantigenic determinants, preferably with spacings of 0.5 to 30nanometers, preferably 5 to 15 nanometers.

Passive immunization: as used herein, the term “passive immunization”refers to the administration, by any route, of exogenously producedimmune molecules (e.g. antibodies) or cells (e.g. T-cells) into ananimal. Passive immunization differs from “active” immunization, whereimmunity is obtained by introduction of an immunogen, vaccine, antigenor hapten-canier conjugate into an individual to elicit an immuneresponse.

Pili: As used herein, the term “pili” (singular being “pilus”) refers toextracellular structures of bacterial cells composed of protein monomers(e.g., pilin monomers) which are organized into ordered and repetitivepatterns. Further, pili are structures which are involved in processessuch as the attachment of bacterial cells to host cell surfacereceptors, inter-cellular genetic exchanges, and cell-cell recognition.Examples of pili include Type-1 pili, P-pili, F1C pili, S-pili, and987P-pili. Additional examples of pili are set out elsewhere herein.

Pilus-like structure: As used herein, the phrase “pilus-like structure”refers to structures having characteristics similar to that of pili andcomposed of protein monomers. One example of a “pilus-like structure” isa structure formed by a bacterial cell which expresses modified pilinproteins that do not form ordered and repetitive arrays that areessentially identical to those of natural pili.

Polypeptide: As used herein the term “polypeptide” refers to a polymercomposed of amino acid residues, generally natural amino acid residues,linked together through peptide bonds. A polypeptide may not necessarilybe limited in size, and include both proteins and peptides. A peptide isa polypeptide of a typical size of about five to about 50 amino acids,or any number amino acids within this general range. A peptide may,however, also be of longer length, for example up to 120-150 aminoacids.

Protein: As used herein, the term protein refers to a polypeptidegenerally of a size of above about 5 or more, 10 or more 20 or more, 25or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more,1000 or more, 2000 or more amino acids. Proteins generally have adefined three dimensional structure although they do not necessarilyneed to, and are often referred to as folded, as opposed to peptides andpolypeptides which often do not possess a defined three-dimensionalstructure, but rather can adopt a large number of differentconformations, and are referred to as unfolded. Peptides may, howeveralso have a defined three-dimensional structure.

Purified: As used herein, when the term “purified” is used in referenceto a molecule, it means that the concentration of the molecule beingpurified has been increased relative to molecules associated with it inits natural environment, or environment in which it was produced, foundor synthesized. Naturally associated molecules include proteins, nucleicacids, lipids and sugars but generally do not include water, buffers,and reagents added to maintain the integrity or facilitate thepurification of the molecule being purified. For example, even if mRNAis diluted with an aqueous solvent during oligo dT columnchromatography, mRNA molecules are purified by this chromatography ifnaturally associated nucleic acids and other biological molecules do notbind to the column and are separated from the subject mRNA molecules.According to this definition, a substance may be 5% or more, 10% ormore, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more,70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 99% ormore, or 100% pure when considered relative to its contaminants.

Receptor: As used herein, the term “receptor” refers to proteins orglycoproteins or fragments thereof capable of interacting with anothermolecule, called the ligand. The ligand may belong to any class ofbiochemical or chemical compounds. The receptor need not necessarily bea membrane-bound protein. Soluble protein, like e.g., maltose bindingprotein or retinol binding protein are receptors as well.

Residue: As used herein, the term “residue” is meant to mean a specificamino acid in a polypeptide backbone or side chain.

Recombinant host cell: As used herein, the term “recombinant host cell”refers to a host cell into which one ore more nucleic acid molecules ofthe invention have been introduced. Host cells include eukaryotesinclude e.g. mammalian, insect, plant, avian, yeast; and prokaryotice.g. E. coli, B. subtilis, etc.

Recombinant virus: As used herein, the phrase “recombinant virus” refersto a virus that is genetically modified by the hand of man. The phrasecovers any virus known in the art. More specifically, the phrase refersto a an alphavirus genetically modified by the hand of man, and mostspecifically, the phrase refers to a Sinbis virus genetically modifiedby the hand of man.

RNA-phage: As used herein, the term “RNA-phage” refers to RNA virusesinfecting bacteria, more specifically to single-stranded positive-senseRNA viruses infecting bacteria.

Vector: As used herein, the term “vector” refers to an agent (e.g., aplasmid or virus) used to transmit genetic material to a host cell. Avector may be composed of either DNA or RNA.

Virus-like particle (VLP): As used herein, the term “virus-likeparticle” refers to a structure resembling a virus particle. Moreover, avirus-like particle in accordance with the invention is non replicativeand noninfectious since it lacks all or part of the viral genome, inparticular the replicative and infectious components of the viralgenome. A virus-like particle in accordance with the invention maycontain nucleic acid distinct from their genome. A typical and preferredembodiment of a virus-like particle in accordance with the presentinvention is a viral capsid such as the viral capsid of thecorresponding virus, bacteriophage, or RNA-phage. The terms “viralcapsid” or “capsid”, as interchangeably used herein, refer to amacromolecular assembly composed of viral protein subunits. Typicallyand preferably, the viral protein subunits assemble into a viral capsidand capsid, respectively, having a structure with an inherent repetitiveorganization, wherein said structure is, typically, spherical ortubular. For example, the capsids of RNA-phages or HBcAg's have aspherical form of icosahedral symmetry. The term “capsid-like structure”as used herein, refers to a macromolecular assembly composed of viralprotein subunits ressembling the capsid morphology in the above definedsense but deviating from the typical symmetrical assembly whilemaintaining a sufficient degree of order and repetitiveness.

Virus-like particle of a bacteriophage: As used herein, the term“virus-like particle of a bacteriophage” refers to a virus-like particleresembling the structure of a bacteriophage, being non replicative andnoninfectious, and lacking at least the gene or genes encoding for thereplication machinery of the bacteriophage, and typically also lackingthe gene or genes encoding the protein or proteins responsible for viralattachment to or entry into the host. This definition should, however,also encompass virus-like particles of bacteriophages, in which theaforementioned gene or genes are still present but inactive, and,therefore, also leading to non-replicative and noninfectious virus-likeparticles of a bacteriophage.

VLP of RNA phage coat protein: The capsid structure formed from theself-assembly of 180 subunits of RNA phage coat protein and optionallycontaining host RNA is referred to as a “VLP of RNA phage coat protein”.A specific example is the VLP of Qβ coat protein. In this particularcase, the VLP of Qβ coat protein may either be assembled exclusivelyfrom Qβ CP subunits (generated by expression of a Qβ CP gene containing,for example, a TAA stop codon precluding any expression of the longer A1protein through suppression, see Kozlovska, T. M., et al., Intervirology39: 9-15 (1996)), or additionally contain A1 protein subunits in thecapsid assembly.

Virus particle: The term “virus particle” as used herein refers to themorphological form of a virus. In some virus types it comprises a genomesurrounded by a protein capsid; others have additional structures (e.g.,envelopes, tails, etc.).

One, a, or an: When the terms “one,” “a,” or “an” are used in thisdisclosure, they mean “at least one” or “one or more,” unless otherwiseindicated.

As used herein when referring to any numerical value, the term “about”means a value of +10% of the stated value (e.g., “about 50° C.”encompasses a range of temperatures from 45° C. to 55° C., inclusive;similarly, “about 100 mM” encompasses a range of concentrations from 90mM to 110 mM inclusive).

Overview

We have now developed potent immunogens for the induction of antibodiesspecific for angiotensin peptides which are effective even without theuse of adjuvants, and which may allow specific targeting ofangotensinogen, angiotensin I or angiotensin II. The immunogens consistof angiotensin peptide moieties which are bound to virus-like particles(VLP) or other core particles such as bacterial pili or pilus-likeparticles. This results in a highly immunogenic repetitive antigen arraywhich is able to stimulate antibody formation even without the use ofadjuvants. Depending on the amino acid sequence of the angiotensinpeptide moieties used, high antibody titers are induced, and, moreover,can be specifically induced against the N- or C-terminal ends ofangiotensinogen, angiotensin I or angiotensin II. This allows thespecific targeting of only one species of angiotensin peptides or acombination thereof. The immunogens of the present invention thus can beused in an immunotherapeutic approach to combat conditions associatedwith elevated levels of angiotensin peptide moieties, particularlyangiotensin II and derivatives thereof, produced by the RAS.

Formation of conjugates of the invention, i.e. binding one or moreangiotensin peptide moieties to the core particle (e.g., the VLP), isachieved by attachment, linkage, fusion or other binding, includingcovalent and non covalent bonds. In one embodiment, the VLP contains afirst attachment site, the organic molecule contains a second attachmentsite. Association between the organic molecule occurs by linking thefirst and second attachment sites directly, or via a third molecule.Attachment sites may occur naturally, or may be introduced.

Immunization of animals with conjugates of angiotensin peptide moietiesand core particles, or with conjugates comprising such conjugates asprovided by the invention, induce a strong immune response against thedisplayed angiotensin peptide moieties. Hence, the conjugates andconjugates of the invention are useful for the stimulation of an immuneresponse against a variety of angiotensin peptide moieties orderivatives thereof, and thus for the use in animals. The presentinvention also relates to a vaccine comprising an immunologicallyeffective amount of one or more of the conjugates or conjugates of thepresent invention together with a pharmaceutically acceptable diluent,carrier or excipient. The conjugates and conjugates of the invention canbe used to vaccinate an animal against one or more angiotensin peptidemoieties or derivatives thereof. The vaccination can be for prophylacticor therapeutic purposes, or both. In a related aspect immune molecules,such as antibodies, generated against such conjugates or conjugates maybe used for treatment, prophylaxis or diagnosis of a disease, conditionor disorder. Such antibodies, conjugates and conjugates of the inventionare also useful as components kits.

Thus, in one aspect the invention provides conjugates of one or moreangiotensin peptide moieties with a carrier in an ordered and repetitiveangiotensin peptide moiety-carrier conjugate, and methods of making suchconjugates. The invention also provides conjugates comprising at leastone such conjugate of the invention and at least one other component,suitably at least one excipient or carrier and particularly at least onepharmaceutically acceptable excipient or carrier. The conjugates andconjugates of the invention are useful for inducing immune responsesagainst angiotensin peptide moieties. Such an immune response can be canbe utilized to generate antibodies, useful for therapeutic, prophylacticand diagnostic purposes.

The conjugates of the present invention comprise highly ordered andrepetitive arrays of one or more angiotensin peptide moieties. Conjugatearrays according to this aspect of the invention comprise (a) a coreparticle, comprising a first attachment site and (b) an angiotensinpeptide moiety comprising a second attachment site, wherein the elements(a) and (b) are associated through the first and second attachment sitesto form said ordered and repetitive arrays of angiotensin peptidemoieties.

Core particles suitably used in the conjugates and conjugates of theinvention may be natural or non-natural. Natural core particles used inthe conjuages and conjugates of the present invention include virusparticles, virus-like particles, and pili. The proteins of these naturalcore particles may be natural or recombinant. The first attachment siteson the core particle may occur naturally or may be introduced viachemical or recombinant means. Angiotensin peptide moieties used in theconjugates and conjugates of the present invention are those suitablefor inducing immune responses against a variety of components of the RAS(i.e., a variety of angiotensin peptide moieties or derivativesthereof), including but not limited to an angiotensin peptide,preferably those comprising, or alternatively consisting of, thesequence, or fragments thereof, of angiotensinogen, angiotensin I (adecapeptide of formula Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) orangiotensin II (an octapeptide of formulaAsp-Arg-Val-Tyr-Ile-His-Pro-Phe), or functionally equivalent variantsthereof including those angiotensin peptide moieties described elsewhereherein. The second attachment site on the angiotensin peptide moiety maynaturally occur or be introduced. The interaction between first andsecond sites may be direct, or may involve at least one other molecule,e.g. a linker. Furthermore, cross-linking molecules may be used inaccordance with the present invention for association of the first andsecond attachment sites. Cross-linking molecules are typically used inaddition to the linker.

The conjugates and conjugates of the invention are suprisingly effectivein inducing immune responses, particularly antibodies, against a varietyof angiotensin peptide moieties. Thus, they are useful in conjugatessuitable for immunization of animals for therapeutic or prophylaxisagainst diseases, disorders or conditions associated with the RAS,including but not limited to hypertension, stroke, infarction,congestive heart failure, kidney failure or retinal hemorrhage.Antibodies produced by immunization with the conjugates and conjugatesof the invention are also useful for therapeutic and prophylacticpurposes.

In other embodiments, the invention provides methods of treatment andprevention of a disease utilizing the conjugates and conjugates of theinvention. In another embodiment, the invention provides kits suitablefor diagnosis and screening.

Conjugates of Ordered and Repetitive Arrays

The present invention provides conjugates, and conjugates of conjugates,comprising an ordered and repetitive array of one or more angiotensinpeptide moieties. Furthermore, the invention conveniently enables thepractitioner to construct ordered and repetitive arrays for variouspurposes, and preferably the induction of an immune response against oneor more angiotensin peptide moieties or derivatives thereof.

Conjugates of the invention essentially comprise, or alternativelyconsist of, two elements: (1) a non-natural molecular scaffold; and (2)at least one angiotensin peptide moiety with at least one secondattachment site capable of association through at least one bond to saidfirst attachment site.

The non-natural molecular scaffold comprises, or alternatively consistsof: (a) a core particle selected from the group consisting of (1) a coreparticle of non-natural origin and (2) a core particle of naturalorigin; and (b) at least one first attachment site connected to saidcore particle by at least one covalent bond. Core particles used in theconjugates, conjugates and methods of the invention include inorganicmolecules, virus particles, virus-like particles, and bacterial pili.The angiotensin peptide moieties used in the conjugates, conjugates andmethods of the invention have at least one second attachment site whichis selected from the group consisting of (a) an attachment site notnaturally occurring within the angiotensin peptide moiety; and (b) anattachment site naturally occurring within the angiotensin peptidemoiety

The invention provides for an ordered and repetitive array through anassociation of the second attachment site to the first attachment siteby way of at least one bond. Thus, the angiotensin peptide moiety andthe non-natural molecular scaffold are brought together through thisassociation of the first and the second attachment site to form anordered and repetitive antigen array.

The practioner may specifically design the angiotensin peptide moietyand the second attachment site such that the arrangement of all themoieties bound to the non-natural molecular scaffold, or in certainembodiments to the core particle, will be uniform. For example, one mayplace a single second attachment site on the angiotensin peptide moiety,thereby ensuring through design that all angiotensin peptide moietiesthat are attached to the non-natural molecular scaffold are positionedin a uniform way. In one such aspect of the invention, one or moreadditional amino acids (leading to a non-naturally occurring secondattachment site) are added either at the C- or at the N-terminus of theangiotensin peptide moiety sequences in order to assure, in particular,an oriented and ordered association with the core particle in accordancewith the present invention. Thus, the invention provides a convenientmeans of placing any angiotensin peptide moiety onto a non-naturalmolecular scaffold in a defined order and in a manner which forms arepetitive pattern.

As will be clear to those of ordinary skill in the art, certainembodiments of the invention involve the use of recombinant nucleic acidtechnologies such as cloning, polymerase chain reaction, thepurification of DNA and RNA, the expression of recombinant proteins inprokaryotic and eukaryotic cells, etc. Such methodologies are well knownto those skilled in the art and may be conveniently found in publishedlaboratory methods manuals (e.g., Sambrook, J. et al., eds., MOLECULARCLONING, A LABORATORY MANUAL, 2nd. edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al.,eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc.(1997)). Fundamental laboratory techniques for working with tissueculture cell lines (Celis, J., ed., CELL BIOLOGY, Academic Press, 2^(nd)edition, (1998)) and antibody-based technologies (Harlow, E. and Lane,D., “Antibodies: A Laboratory Manual,” Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1988); Deutscher, M. P., “Guide to ProteinPurification,” Meth. Enzymol. 128, Academic Press San Diego (1990);Scopes, R. K., “Protein Purification Principles and Practice,” 3^(rd)ed., Springer-Verlag, New York (1994)) are also adequately described inthe literature, all of which are incorporated herein by reference.

Furthermore, technologies for coupling organic molecules to amino acidsand means for making derivatives of angiotensin peptide moietiescontaining appropriate second attachment sites such as are neccessaryfor the practice of the invention are well known to those of skill inthe art. Such methodologies may be found in chemical text books andpublications, examples of which are included below and are incorportatedby reference; U.S. Pat. No. 5,876,727; WO 99/61054; Isomura, S. et al.J. Org. Chem. 66:4115-4121 (2001); Matsushita, H. et al. Biochem.Biophys. Res. Comm. 57:1006-1010. (1974); Langone, J. L. and VanVunakis, H., Methods Enzymol. 84:628-640 (1982); Wong, Chemistry ofProtein Conjugation and Cross-Linking. CRC Press, Inc., Boca Raton, Fla.(1991.)

Core Particles and Non-Natural Molecular Scaffolds

In one embodiment, the present invention provides methods for theformation of an ordered and repetitive array of one or more angiotensinpeptide moieties. By the invention, this occurs by the association of acore particle to which is attached one or more angiotensin peptidemoieties via first and second attachment sites.

Thus, one element in certain conjugates and conjugates of the inventionis a non-natural molecular scaffold comprising, or alternativelyconsisting of, a core particle and a first attachment site. Morespecifically, the non-natural molecular scaffold comprises, oralternatively consists of, (a) a core particle of natural or non-naturalorigin and (b) at least one first attachment site connected to the coreparticle by at least one covalent bond.

Core particles. In one embodiment of the present invention, a coreparticle is a synthetic polymer, a lipid micelle or a metal. Such coreparticles are known in the art, providing a basis from which to buildthe novel non-natural molecular scaffold of the invention. By way ofexample, synthetic polymer or metal core particles are disclosed in U.S.Pat. No. 5,770,380, and U.S. Pat. No. 5,334,394, which are incorporatedby reference herein in their entirities. Suitable metals include, butare not limited to, chromium, rubidium, iron, zinc, selenium, nickel,gold, silver, platinum. Suitable ceramic materials include, but are notlimited to, silicon dioxide, titanium dioxide, aluminum oxide, rutheniumoxide and tin oxide. The core particles of this embodiment may be madefrom organic materials including, but not limited to, carbon andsuitable polymers, including polystyrene, nylon and nitrocellulose. Fornanocrystalline particles, particles made from tin oxide, titaniumdioxide or carbon (diamond) are useful. Lipid micelles for use in thepresent invention are prepared by any means known in the art, forexample, Baiselle and Millar (Biophys. Chem. 4:355-361 (1975)) or Cortiet al. (Chem. Phys. Lipids 38:197-214 (1981)) or Lopez et al. (FEBSLett. 426:314-318 (1998)) or Topchieva and Karezin (J. Colloid InterfaceSci. 213:29-35 (1999)) or Morein et al., (Nature 308:457-460 (1984)),which are incorporated herein by reference in their entirities.

In one embodiment of the invention the core particle is produced througha biological process, which may be natural or non-natural. For example,viruses and bacterial pili or pilus-like structures are formed fromproteins which are organized into ordered and repetitive structures.Therefore, the present invention comprises conjugates, conjugates andmethods comprising useful core particles which include, but are notlimited to a virus, virus-like particle, a bacterial pilus, a phage, aviral capsid particle or fragments thereof. In certain such embodiments,the proteins may be recombinant.

In certain embodiments, the core particle of the non-natural molecularscaffold comprises a virus, a bacterial pilus, a structure formed frombacterial pilin, a bacteriophage, a virus-like particle, a viral capsidparticle or a recombinant form thereof. Any virus known in the arthaving an ordered and repetitive coat and/or core protein structure maybe selected for use as in the methods, conjugates and conjugates of theinvention as a non-natural molecular scaffold. Examples of suitableviruses include, but are not limited to, sindbis and other alphaviruses,rhabdoviruses (e.g. vesicular stomatitis virus), picornaviruses (e.g.,human rhino virus, Aichi virus), togaviruses (e.g., rubella virus),orthomyxoviruses (e.g., Thogoto virus, Batken virus, fowl plague virus),polyomaviruses (e.g., polyomavirus BK, polyomavirus JC, avianpolyomavirus BFDV), parvoviruses, rotaviruses, bacteriophage Qβ,bacteriophage R17, bacteriophage M11, bacteriophage MX1, bacteriophageNL95, bacteriophage fr, bacteriophage GA, bacteriophage SP,bacteriophage MS2, bacteriophage f2, bacteriophage PP7, bacteriophageAP205, Norwalk virus, foot and mouth disease virus, a retrovirus,Hepatitis B virus, Tobacco mosaic virus, Flock House Virus, and humanPapillomavirus (for example, see Table 1 in Bachman, M. F. andZinkernagel, R. M., Immunol. Today 17:553-558 (1996)). In more specificexemplary embodiments of the present invention the core particle maycomprise, or alternatively consist of, recombinant proteins ofRotavirus, recombinant proteins of Norwalk virus, recombinant proteinsof Alphavirus, recombinant proteins which form bacterial pili orpilus-like structures, recombinant proteins of Foot and Mouth Diseasevirus, recombinant proteins of Retrovirus, recombinant proteins ofHepatitis B virus (e.g., a HBcAg), recombinant proteins of Tobaccomosaic virus, recombinant proteins of Flock House Virus, and recombinantproteins of human Papillomavirus.

The core particle used in conjugates, conjugates and methods of theinvention may further comprise, or alternatively consist of, one or morefragments of such proteins, as well as variants of such proteins whichretain the ability to associate with each other to form ordered andrepetitive antigen or antigenic determinant arrays. For example, asexplained in commonly owned copending U.S. patent application Ser. No.10/050,902, (filed Jan. 18, 2002, the disclosure of which isincorporated herein by reference in its entirety) core particles may beformed from variant forms of the human HBcAg which differ markedly fromthe wild-type particle in amino acid sequence identity and similarity,and in sequence length. For example, amino acid sequence of the HBcAg ofHepatitis B viruses which infect snow geese and ducks differssufficiently from that of HBcAg of viruses infected mammals thatalignment of the proteins is difficult. However, both viruses retain theability to form core structures suitable for the formation of orderedrepetitive antigen arrays. Similarly, HBcAg may retain the ability toform multimeric particles, typical of a virus, after removal ofN-terminal leader sequences, further deletions, substitutions, oradditions to the sequence. Methods which can be used to determinewhether proteins form such structures comprise gel filtration, agarosegel electrophoresis, sucrose gradient centrifugation and electronmicroscopy (e.g., Koschel, M. et al., J. Virol 73: 2153-2160 (1999)).

First Attachment Sites. Whether natural or non-natural, the coreparticle used in the conjugates, conjugates and methods of the presentinvention will generally possess a component comprising a firstattachment site that is attached to the natural or non-natural coreparticle by at least one covalent bond. The element comprising the firstattachment site is bound to a core particle in a non-random fashion thatprovides a nucleation site for creating an ordered and repetitive array.Ideally, but not necessarily, this element is associated with the coreparticle in a geometric order. The first attachment site may be anatural part of the core particle, such as a surface exposed amino acidresidue suitable for coupling to the second attachment site. Forexample, lysine and cysteine may form non-peptide bonds via reactivegroups on the amino acid. Alternatively, an element containing the firstattachment site may be introduced into the core particle via chemicalcoupling or through the design of recombinant molecules. The firstattachment site may be, or be found on, any element comprising bound toa core particle by at least one covalent bond.

The first attachment site may comprise, or alternatively may consist of,a protein, a polypeptide, a peptide, an amino acid (i.e., a residue of aprotein, a polypeptide or peptide), a sugar, a polynucleotide, a naturalor synthetic polymer, a secondary metabolite or compound (biotin,fluorescein, retinol, digoxigenin, metal ions,phenylmethylsulfonylfluoride), or a combination thereof, or a chemicallyreactive group thereof. In a more specific embodiment, the firstattachment site comprising an antigen, an antibody or antibody fragment,biotin, avidin, strepavidin, a receptor, a receptor ligand, a ligand, aligand-binding protein, an interacting leucine zipper polypeptide, anamino group, a chemical group reactive to an amino group; a carboxylgroup, chemical group reactive to a carboxyl group, a sulfhydryl group,a chemical group reactive to a sulfhydryl group, or a combinationthereof.

In one embodiment, the invention utilizes genetic engineering of a virusto create a fusion between an ordered and repetitive viral envelopeprotein the element comprising the first attachment site whichcomprising a heterologous protein, peptide, antigenic determinant or areactive amino acid residue of choice. Other genetic manipulations knownto those in the art may be included in the construction of thenon-natural molecular scaffold; for example, it may be desirable torestrict the replication ability of the recombinant virus throughgenetic mutation. The viral protein selected for fusion to the proteincontaining the first attachment site protein should have an organizedand repetitive structure. Such an organized and repetitive structureinclude paracrystalline organizations with a spacing of 0.5-30 nm,preferably of 5-15 nm on the surface of the virus. The creation of thistype of fusion protein will result in multiple, ordered and repetitivefirst attachment sites on the surface of the virus. Thus, the orderedand repetitive organization of the first attachment sites resultingtherefrom will reflect the normal organization of the native viralprotein.

As will be understood by those of ordinary skill in the art, the firstattachment site may be or be a part of any suitable protein,polypeptide, sugar, polynucleotide, peptide (amino acid), natural orsynthetic polymer, a secondary metabolite or combination thereof thatmay serve to specifically attach the antigen or antigenic determinant ofchoice to the non-natural molecular scaffold. In one embodiment, theattachment site is a protein or peptide that may be selected from thoseknown in the art. For example, the first attachment site may be aligand, a receptor, a lectin, avidin, streptavidin, biotin, an epitopesuch as an HA or T7 tag, Myc, Max, immunoglobulin domains and any otheramino acid sequence known in the art that would be useful as a firstattachment site.

It will be further understood by those of ordinary skill in the art thatin another embodiment of the invention, the first attachment site may becreated secondarily to the creation of an element carrying the firstattachment site (e.g., protein or polypeptide) utilized in constructingthe in-frame fusion to the capsid protein. For example, a protein may beutilized for fusion to the envelope protein with an amino acid sequenceknown to be glycosylated in a specific fashion, and the sugar moietyadded as a result may then serve at the first attachment site of theviral scaffold by way of binding to a lectin serving as the secondaryattachment site of an antigen. Alternatively, a sequence may bebiotinylated in vivo and the biotin moiety may serve as the firstattachment site of the invention, or the sequence may be subjected tochemical modification of distinct amino acid residues in vitro, themodification serving as the first attachment site.

In one specific embodiment of the invention, the first attachment siteis the JUN-FOS leucine zipper protein domain that is fused in frame tothe Hepatitis B capsid (core) protein (HBcAg). However, it will be clearto those of ordinary skill in the art that other viral capsid proteinsmay be utilized in the fusion protein construct for locating the firstattachment site in the non-natural molecular scaffold of the invention.For example, in other embodiments of the invention, the first attachmentsite is selected to be a lysine or cysteine residue that is fused inframe to the HBcAg. It will also be clear to all individuals in the artthat other viral capsid or virus-like particles may be utilized in thefusion protein construct for locating the first attachment in thenon-natural molecular scaffold of the invention.

Viral particles. In one embodiment of the invention, the non-naturalmolecular scaffold is a recombinant alphavirus, and more specifically, arecombinant Sindbis virus. Several members of the alphavirus family,Sindbis (Xiong, C. et al., Science 243:1188-1191 (1989); Schlesinger,S., Trends Biotechnol. 11:18-22 (1993)), Semliki Forest Virus (SFV)(Liljeström, P. & Garoff, H., Bio/Technology 9:1356-1361 (1991)) andothers (Davis, N. L. et al., Virology 171:189-204 (1989)), have receivedconsiderable attention for use as virus-based expression vectors for avariety of different proteins (Lundstrom, K., Curr. Opin. Biotechnol.8:578-582 (1997); Liljeström, P., Curr. Opin. Biotechnol. 5:495-500(1994)) and as candidates for vaccine development. The use ofalphaviruses for the expression of heterologous proteins and thedevelopment of vaccines has been disclosed (see U.S. Pat. Nos.5,766,602; 5,792,462; 5,739,026; 5,789,245; and 5,814,482; thedisclosures all of which are incorporated by reference in theirentirities). The construction of an alphaviral scaffold according tothis aspect of the invention may be done by means generally known in theart of recombinant DNA technology, as described by the aforementionedarticles, which are incorporated herein by reference. A variety ofrecombinant host cells can be utilized to produce a viral-based coreparticle for attachment of one or more angiotensin peptide moieties.

Packaged RNA sequences can also be used to infect host cells. Thesepackaged RNA sequences can be introduced to host cells by adding them tothe culture medium. For example, the preparation of non-infectivealpahviral particles is described in a number of sources, including“Sindbis Expression System”, Version C (Invitrogen Corporation, CarlsbadCalif.; Catalog No. K750-1).

When mammalian cells are used as recombinant host cells for theproduction of viral-based core particles, these cells will generally begrown in tissue culture. Methods for growing cells in culture are wellknown in the art (see, e.g., Celis, J., ed., CELL BIOLOGY, AcademicPress, 2^(nd) edition, (1998); Sambrook, J. et al., eds., MOLECULARCLONING, A LABORATORY MANUAL, 2nd. edition, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al.,eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc.(1997); Freshney, R., CULTURE OF ANIMAL CELLS, Alan R. Liss, Inc.(1983)).

The invention thus includes viral-based core particles which comprise,or alternatively consist of, a virus, virus-like particle, a phage, aviral capsid particle or a recombinant form thereof. Ordinarily skilledartisans have the knowledge to produce such core particles and attachfirst attachment sites thereto. The production of Hepatitis B virus-likeparticles, in particular those assembled or self-assembled from HBcAg,and measles viral capsid particles as core particles is disclosed inExamples 17 to 22 of WO 00/32227, which is explicitly incorporatedherein by reference. In such embodiments, the JUN leucine zipper proteindomain or FOS leucine zipper protein domain may be used as a firstattachment site for the non-natural molecular scaffold of the invention.One of ordinary skill in the art will be aware of methods forconstructing Hepatitis B core particles carrying an in-frame fusedpeptide with a reactive lysine residue and angiotensin peptide moietiescarrying a genetically fused cysteine residue, as first and secondattachment site, respectively.

In other embodiments, the core particles used in conjugates of theinvention are composed of a Hepatitis B capsid (core) protein (HBcAg), afragment of a HBcAg, or other protein or peptide which can formvirus-like particles, which are ordered arrays, which have been modifiedto either eliminate or reduce the number of free cysteine residues. Zhouet al. (J. Virol. 66:5393-5398 (1992)) demonstrated that HBcAgs whichhave been modified to remove the naturally resident cysteine residuesretain the ability to associate and form multimeric structures. Thus,core particles suitable for use in conjugates of the invention includethose comprising modified HBcAgs, or fragments thereof, in which one ormore of the naturally resident cysteine residues have been eitherdeleted or substituted with another amino acid residue (e.g., a serineresidue). In one embodiment of the invention, a modified HBcAgcomprising the amino acid sequence shown in SEQ ID NO:1, or subportionthereof, is used to prepare non-natural molecular scaffolds. Inparticular, modified HBcAgs suitable for use in the practice of theinvention include proteins in which one or more of the cysteine residuesat positions corresponding to positions 48, 61, 107 and 185 of a proteinhaving the amino acid sequence shown in SEQ ID NO:1 have been eitherdeleted or substituted with other amino acid residues (e.g., a serineresidue). As one skilled in the art would recognize, cysteine residuesat similar locations in HBcAg variants having amino acids sequenceswhich differ from that shown in SEQ ID NO:1 could also be deleted orsubstituted with other amino acid residues. The modified HBcAg variantscan then be used to prepare vaccine conjugates of the invention.

Under certain circumstances (e.g., when a heterobifunctionalcross-linking reagent is used to attach one or more angiotensin peptidemoieties to the non-natural molecular scaffold), the presence of freecysteine residues in the HBcAg is believed to lead to covalent couplingof toxic components to core particles, as well as the cross-linking ofmonomers to form undefined species. Further, in many instances, thesetoxic components may not be detectable with assays performed onconjugates of the invention. This is so because covalent coupling oftoxic components to the non-natural molecular scaffold would result inthe formation of a population of diverse species in which toxiccomponents are linked to different cysteine residues, or in some casesno cysteine residues, of the HBcAgs. In other words, each free cysteineresidue of each HBcAg will not be covalently linked to toxic components.Further, in many instances, none of the cysteine residues of particularHBcAgs will be linked to toxic components. Thus, the presence of thesetoxic components may be difficult to detect because they would bepresent in a mixed population of molecules. The administration to anindividual of HBcAg species containing toxic components, however, couldlead to a potentially serious adverse reaction.

It is well known in the art that free cysteine residues can be involvedin a number of chemical side reactions. These side reactions includedisulfide exchanges, reaction with chemical substances or metabolitesthat are, for example, injected or formed in a combination therapy withother substances, or direct oxidation and reaction with nucleotides uponexposure to UV light. Toxic adducts could thus be generated, especiallyconsidering the fact that HBcAgs have a strong tendency to bind nucleicacids. Detection of such toxic products in antigen-capsid conjugateswould be difficult using capsids prepared using HBcAgs containing freecysteines and heterobifunctional cross-linkers, since a distribution ofproducts with a broad range of molecular weight would be generated. Thetoxic adducts would thus be distributed between a multiplicity ofspecies, which individually may each be present at low concentration,but reach toxic levels when together.

In view of the above, one advantage to the use of HBcAgs in vaccineconjugates which have been modified to remove naturally residentcysteine residues is that sites to which toxic species can bind whenantiogensin peptide moieties are attached to the non-natural molecularscaffold would be reduced in number or eliminated altogether. Further, ahigh concentration of cross-linker can be used to produce highlydecorated particles without the drawback of generating a plurality ofundefined cross-linked species of HBcAg monomers (i.e., a diversemixture of cross-linked monomeric HbcAgs).

A number of naturally occurring HBcAg variants suitable for use in thepractice of the present invention have been identified. Yuan et al., (J.Virol. 73:10122-10128 (1999)), for example, describe variants in whichthe isoleucine residue at position corresponding to position 97 in SEQID NO:1 is replaced with either a leucine residue or a phenylalanineresidue. The amino acid sequences of a number of HBcAg variants, as wellas several Hepatitis B core antigen precursor variants, are disclosed inGenBank reports AAF121240, AF121239, X85297, X02496, X85305, X85303,AF151735, X85259, X85286, X85260, X85317, X85298, AF043593, M20706,X85295, X80925, X85284, X85275, X72702, X85291, X65258, X85302, M32138,X85293, X85315, U95551, X85256, X85316, X85296, AB033559, X59795, X8529,X85307, X65257, X85311, X85301, X85314, X85287, X85272, X85319,AB010289, X85285, AB010289, AF121242, M90520, P03153, AF110999, andM95589, the disclosures of each of which are incorporated herein byreference. These HBcAg variants differ in amino acid sequence at anumber of positions, including amino acid residues which corresponds tothe amino acid residues located at positions 12, 13, 21, 22, 24, 29, 32,33, 35, 38, 40, 42, 44, 45, 49, 51, 57, 58, 59, 64, 66, 67, 69, 74, 77,80, 81, 87, 92, 93, 97, 98, 100, 103, 105, 106, 109, 113, 116, 121, 126,130, 133, 135, 141, 147, 149, 157, 176, 178, 182 and 183 in SEQ ID NO:1.

Further HBcAg variants suitable for use in the compositions of theinvention, and which may be further modified according to the disclosureof this specification are described in WO 00/198333, WO 00/177158 and WO00/214478, herein included by reference in their entirety.

HBcAgs suitable for use in the present invention may be derived from anyorganism so long as they are able to associate to form an ordered andrepetitive antigen array. Generally processed HBcAgs (i.e., those whichlack leader sequences) will be used in the vaccine conjugates of theinvention. The present invention includes vaccine conjugates, as well asmethods for using these conjugates, which employ the above describedvariant HBcAgs for the preparation of non-natural molecular scaffolds.Further included within the scope of the invention are additional HBcAgvariants which are capable of associating to form dimeric or multimericstructures. Thus, the invention further includes vaccine conjugatescomprising HBcAg polypeptides comprising, or alternatively consistingof, amino acid sequences which are at least about 80%, about 85%, about90%, about 95%, about 97%, or about 99% identical to any of the aminoacid sequences shown in the above sequences, including SEQ ID No: 1, andforms of these proteins which have been processed, where appropriate, toremove the N-terminal leader sequence.

Whether the amino acid sequence of a polypeptide has an amino acidsequence that is at least about 80%, about 85%, about 90%, about 95%,about 97%, or about 99% identical to one of the amino acid sequencesshown above, or a subportion thereof, can be determined conventionallyusing known computer programs such the Bestfit program. When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, about 95% identical to a referenceamino acid sequence according to the present invention, the parametersare set such that the percentage of identity is calculated over the fulllength of the reference amino acid sequence and that gaps in homology ofup to 5% of the total number of amino acid residues in the referencesequence are allowed. In such a manner, comparisons may be made betweenthe amino acid sequence of HBcAg of SEQ ID NO:1 and other HBcAg. Whencomparing proteins that are relatively similar, reference to an aminoacid residue of a HBcAg variant located at a position which correspondsto a particular position in SEQ ID NO:1, refers to the amino acidresidue which is present at that position in the amino acid sequenceshown in SEQ ID NO:1. The homology between these HBcAg variants is forthe most part high enough among Hepatitis B viruses that infect mammalsso that one skilled in the art would have little difficulty reviewingboth the amino acid sequence shown in SEQ ID NO:1 and that of aparticular HBcAg variant and identifying “corresponding” amino acidresidues. For example, in comparisons between the SEQ ID NO:1 and theamino acid sequence of the an HBcAg derived from a virus which infectswoodchucks, it is readily apparent that a three amino acid residueinsert is present in that sequence between amino acid residues 155 and156 of SEQ ID NO:1.

However, where alignment is difficult, one skilled in the art wouldrecognize the importance of particular amino acids or motifs in asequence. For example, the amino acid sequence of HBcAg from humanviruses differs from duck viruses such that alignment is difficult, yetone skilled in the art would recognize conserved cysteine residues couldbe either substituted with another amino acid residue or deleted priorto their inclusion in vaccine conjugates of the invention.

In one embodiment, the cysteine residues at positions 48 and 107 of aprotein having the amino acid sequence shown in SEQ ID NO: 1 are deletedor substituted with another amino acid residue but the cysteine atposition 61 is left in place. Further, the modified polypeptide is thenused to prepare vaccine conjugates of the invention.

The preparation of preferred Hepatitis B virus-like particles, which canbe used for the present invention, is disclosed, for example, in WO00/32227, and hereby in particular in Examples 17 to 19 and 21 to 24, aswell as in WO 01/85208, and hereby in particular in Examples 17 to 19,21 to 24, 31 and 41, and in pending U.S. application Ser. No. 10/050,902filed by the present assignee on Jan. 18, 2002. For the latterapplication, it is in particular referred to Example 23, 24, 31 and 51.All three documents are explicitly incorporated herein by reference.

As set out in Example 31 of U.S. application Ser. No. 10/050,902 filedby the present assignee on Jan. 18, 2002, the cysteine residues atpositions 48 and 107, which are accessible to solvent, may be removed,for example, by site-directed mutagenesis. In one such example, it hasbeen found that the Cys-48-Ser, Cys-107-Ser HBcAg double mutantconstructed as described in copending U.S. patent application Ser. No.10/050,902, filed Jan. 18, 2002, (which is incorporated herein byreference in its entirety) can be expressed in E. coli.

As discussed above, the elimination of free cysteine residues reducesthe number of sites where toxic components can bind to the HBcAg, andalso eliminates sites where cross-linking of lysine and cysteineresidues of the same or of neighboring HBcAg molecules can occur. Thecysteine at position 61, which is involved in dimer formation and formsa disulfide bridge with the cysteine at position 61 of another HBcAg,will normally be left intact for stabilization of HBcAg dimers andmultimers of the invention. Cross-linking experiments performed with (1)HBcAgs containing free cysteine residues and (2) HBcAgs whose freecysteine residues have been made unreactive with iodacetamide, indicatethat free cysteine residues of the HBcAg are responsible forcross-linking between HBcAgs through reactions betweenheterobifunctional cross-linker derivatized lysine side chains, and freecysteine residues. It was also found that that cross-linking of HBcAgsubunits leads to the formation of high molecular weight species ofundefined size which can not be resolved by SDS-polyacrylamide gelelectrophoresis.

When an angiotensin peptide moiety is linked to the non-naturalmolecular scaffold through a lysine residue, it may be advantageous toeither substitute or delete one or both of the naturally resident lysineresidues located at positions corresponding to positions 7 and 96 in SEQID NO:1, as well as other lysine residues present in HBcAg variants. Theelimination of these lysine residues results in the removal of bindingsites for angiotensin peptide moieties which could disrupt the orderedarray and should improve the quality and uniformity of the final vaccineconjugate.

In many instances, when both of the naturally resident lysine residuesat positions corresponding to positions 7 and 96 in SEQ ID NO:1 areeliminated, another lysine will be introduced into the HBcAg as anattachment site for an angiotensin peptide moiety. Methods for insertingsuch a lysine residue are set out, for example, in copending U.S. patentapplication Ser. No. 10/050,902, filed Jan. 18, 2002, and hereby inparticular in Example 23 of of U.S. application Ser. No. 10/050,902(which is incorporated herein by reference in its entirety). It willoften be advantageous to introduce a lysine residue into the HBcAg when,for example, both of the naturally resident lysine residues at positionscorresponding to positions 7 and 96 in SEQ ID NO:1 are altered and oneseeks to attach the angiotensin peptide moiety to the non-naturalmolecular scaffold using a heterobifunctional cross-linking agent.

The C-terminus of the HBcAg has been shown to direct nuclearlocalization of this protein (Eckhardt et al., J. Virol. 65:575-582(1991).) Further, this region of the protein is also believed to conferupon the HBcAg the ability to bind nucleic acids.

In some embodiments, vaccine conjugates of the invention will containHBcAgs which have nucleic acid binding activity (e.g., which contain anaturally resident HBcAg nucleic acid binding domain). HBcAgs containingone or more nucleic acid binding domains are useful for preparingvaccine conjugates which exhibit enhanced T-cell stimulatory activity.Thus, the vaccine conjugates of the invention include conjugates whichcontain HBcAgs having nucleic acid binding activity. Further includedare vaccine conjugates, as well as the use of such conjugates invaccination protocols, where HBcAgs are bound to nucleic acids. TheseHBcAgs may bind to the nucleic acids prior to administration to anindividual or may bind the nucleic acids after administration.

Further HBcAgs suitable for use in the practice of the present inventioninclude N- and C-terminal truncation mutants, and muteins whose aminoacid sequences comprises or alternatively consists of, amino acidsequences which are at least about 80%, about 85%, about 90%, about 95%,about 97%, or about 99% identical to the above described truncationmutants.

As discussed above, in certain embodiments of the invention, a lysineresidue is introduced as a first attachment site into a polypeptidewhich forms the non-natural molecular scaffold. In preferredembodiments, vaccine conjugates of the invention are prepared using aHBcAg comprising, or alternatively consisting of, amino acids 1-144 oramino acids 1-149 or amino acids 1-185 of SEQ ID NO:1 which is modifiedso that the amino acids corresponding to positions 79 and 80 arereplaced with a peptide having the amino acid sequence ofGly-Gly-Lys-Gly-Gly and the cysteine residues at positions 48 and 107are either deleted or substituted with another amino acid residue, whilethe cysteine at position 61 is left in place.

The invention further includes vaccine conjugates comprising fragmentsof a HBcAg comprising, or alternatively consisting of, an amino acidsequence other than that shown in SEQ ID NO:1 from which a cysteineresidue not present at corresponding location in SEQ ID NO:1 has beendeleted.

Vaccine conjugates of the invention may comprise mixtures of differentHBcAgs. Thus, these vaccine conjugates may be composed of HBcAgs whichdiffer in amino acid sequence. For example, vaccine conjugates could beprepared comprising a “wild-type” HBcAg and a modified HBcAg in whichone or more amino acid residues have been altered (e.g., deleted,inserted or substituted).

The invention further includes vaccine conjugates where the non-naturalmolecular scaffold is prepared using a HBcAg fused to another protein.As discussed above, one example of such a fusion protein is a HBcAg/FOSfusion. Other examples of HBcAg fusion proteins suitable for use invaccine conjugates of the invention include fusion proteins where anamino acid sequence has been added which aids in the formation and/orstabilization of HBcAg dimers and multimers. This additional amino acidsequence may be fused to the C-terminus of the HBcAg. One example, ofsuch a fusion protein is a fusion of a HBcAg with the GCN4 helix regionof Saccharomyces cerevisiae, which forms homodimers via non-covalentinteractions which can be used to prepare and stabilize HBcAg dimers andmultimers.

In one embodiment, the invention provides vaccine conjugates preparedusing HBcAg fusions proteins comprising a HBcAg, or fragment thereof,with a GCN4 polypeptide(PAALKRARNEAARRSRARKLQRMKQLEDKVEELLSKNYHLENEVARLKK) fused to theC-terminus. This GCN4 polypeptide may also be fused to the N-terminus ofthe HbcAg.

HBcAg/src homology 3 (SH3) domain fusion proteins could also be used toprepare vaccine conjugates of the invention. SH3 domains are relativelysmall domains found in a number of proteins which confer the ability tointeract with specific proline-rich sequences in protein bindingpartners (see McPherson, Cell Signal 11:229-238 (1999). HBcAg/SH3 fusionproteins could be used in several ways. First, the SH3 domain could forma first attachment site which interacts with a second attachment site ofthe angiotensin peptide moiety. Similarly, a proline rich amino acidsequence could be added to the HBcAg and used as a first attachment sitefor an SH3 domain second attachment site of an angiotensin peptidemoiety. Second, the SH3 domain could associate with proline rich regionsintroduced into HBcAgs. Thus, SH3 domains and proline rich SH3interaction sites could be inserted into either the same or differentHBcAgs and used to form and stabilized dimers and multimers of theinvention.

As evidenced by the aforementioned example, one of skill in the artwould know how to form a molecular scaffold comprising core particlesand a first attachment site from HBcAg and HBcAg-derived muteins. Byapplication of art-known techniques and routine experimentation, itwould be understood by one of ordinary skill how other viruses could besimilarly used to construct a molecular scaffold.

As presented elsewhere herein, viral capsids may be used for (1) thepresentation of one or more angiotensin peptide moieties and (2) thepreparation of vaccine conjugates of the invention. Particularly, usefulin the practice of the invention are viral capsid proteins, alsoreferred to herein as “coat proteins,” which upon expression formcapsids or capsid-like structures. Thus, these capsid proteins can formcore particles and non-natural molecular scaffolds. Generally, thesecapsids or capsid-like structures form ordered and repetitive arrayswhich can be used for the presentation of antigenic determinants and thepreparation of vaccine conjugates of the invention.

One or more (e.g., one, two, three, four, five, etc.) angiotensinpeptide moieties may be attached by any number of means to one or more(e.g., one, two, three, four, five, etc.) proteins which form viralcapsids or capsid-like structures (e.g., bacteriophage coat proteins),as well as other proteins. For example, angiotensin peptide moieties maybe attached to core particles using first and second attachment sites.Further, one or more (e.g., one, two, three, four, five, etc.)heterobifunctional crosslinkers can be used to attach one or moreangiotensin peptide moieties to one or more proteins which form viralcapsids or capsid-like structures.

Viral capsid proteins, or fragments thereof may be used, for example, toprepare core particles and vaccine conjugates of the invention.Bacteriophage Qβ coat proteins, for example, can be expressedrecombinantly in E. coli. Further, upon such expression these proteinsspontaneously form capsids, which are virus-like particles.Additionally, these capsids form ordered and repetitive antigen arrayswhich can be used in presentation of angiotensin peptide moieties andthe preparation of vaccine conjugates.

In a preferred embodiment, the virus-like particle comprises, consistsessentially of, or alternatively consists of recombinant proteins, orfragments thereof, of a RNA-phage. Preferably, the RNA-phage is selectedfrom the group consisting of a) bacteriophage Qβ; b) bacteriophage R17;c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f)bacteriophage MS2; g) bacteriophage M11; h) bacteriophage MX1; i)bacteriophage NL95; k) bacteriophage f2; l) bacteriophage PP7, and m)bacteriophage AP205.

In another preferred embodiment of the present invention, the virus-likeparticle comprises, or alternatively consists essentially of, oralternatively consists of recombinant proteins, or fragments thereof, ofthe RNA-bacteriophage Qβ or of the RNA-bacteriophage fr or of theRNA-bacteriophage AP205.

Specific examples of bacteriophage coat proteins which can be used toprepare conjugates of the invention include the coat proteins of RNAbacteriophages such as bacteriophage Qβ (SEQ ID NO:3; PIR Database,Accession No. VCBPQβ referring to Qβ CP and SEQ ID NO: 4; Accession No.AAA16663 referring to Qβ A1 protein), bacteriophage R17 (PIR AccessionNo. VCBPR7), bacteriophage fr (PIR Accession No. VCBPFR), bacteriophageGA (GenBank Accession No. NP-040754), bacteriophage SP (GenBankAccession No. CAA30374 referring to SP CP and Accession No. referring toSP A1 protein), bacteriophage MS2 (PIR Accession No. VCBPM2),bacteriophage M11 (GenBank Accession No. AAC06250), bacteriophage MX1(GenBank Accession No. AAC14699), bacteriophage NL95 (GenBank AccessionNo. AAC14704), bacteriophage f2 (GenBank Accession No. P03611),bacteriophage PP7, bacteriophage AP205 (SEQ ID NO:11). As one skilled inthe art would recognize, any protein which forms capsids or capsid-likestructures can be used for the preparation of vaccine conjugates of theinvention. Furthermore, the A1 protein of bacteriophage Qβ (Genbankaccession No. AAA16663 (SEQ ID NO: 4)) or C-terminal truncated formsmissing as much as about 100, about 150 or about 180 amino acids fromits C-terminus may be incorporated in a capsid assembly of Qβ coatproteins. The A1 protein may also be fused an element containing a firstattachment site, for attachment of angiotensin peptide moietiescontaining a second attachment site. Generally, the percentage of A1protein relative to Qβ CP in the capsid assembly will be limited, inorder to insure capsid formation.

Qβ coat protein has also been found to self-assemble into capsids whenexpressed in E. coli (Kozlovska T M. et al., GENE 137: 133-137 (1993)).The obtained capsids or virus-like particles showed an icosahedralphage-like capsid structure with a diameter of 25 nm and T=3 quasisymmetry. Further, the crystal structure of phage Qβ has been solved.The capsid contains 180 copies of the coat protein, which are linked incovalent pentamers and hexamers by disulfide bridges (Golmohammadi, R.et al., Structure 4: 543-5554 (1996)). Other RNA phage coat proteinshave also been shown to self-assemble upon expression in a bacterialhost (Kastelein, R A. et al., Gene 23: 245-254 (1983), Kozlovskaya, T M.et al., Dokl. Akad. Nauk SSSR 287: 452-455 (1986), Adhin, M R. et al.,Virology 170: 238-242 (1989), Ni, CZ., et al., Protein Sci. 5: 2485-2493(1996), Priano, C. et al., J. Mol. Biol. 249: 283-297 (1995)). The Qβphage capsid contains, in addition to the coat protein, the so calledread-through protein A1 and the maturation protein A2. A1 is generatedby suppression at the UGA stop codon and has a length of 329 aa. Thecapsid of phage Qβ recombinant coat protein used in the invention isdevoid of the A2 lysis protein, and contains RNA from the host. The coatprotein of RNA phages is an RNA binding protein, and interacts with thestem loop of the ribosomal binding site of the replicase gene acting asa translational repressor during the life cycle of the virus. Thesequence and structural elements of the interaction are known(Witherell, G W. & Uhlenbeck, O C. Biochemistry 28: 71-76 (1989); Lim F.et al., J. Biol. Chem. 271: 31839-31845 (1996)). The stem loop and RNAin general are known to be involved in the virus assembly (Golmohammadi,R. et al., Structure 4: 543-5554 (1996).)

Upon expression in E. coli, the N-terminal methionine of Qβ coat proteinis usually removed, as we observed by N-terminal Edman sequencing asdescribed in Stoll, E. et al. J. Biol. Chem. 252:990-993 (1977). VLPcomposed from Qβ coat proteins where the N-terminal methionine has notbeen removed, or VLPs comprising a mixture of Qβ coat proteins where theN-terminal methionine is either cleaved or present are also within thescope of the present invention. In a further preferred embodiment of thepresent invention, the virus-like particle comprises, or alternativelyessentially consists of, or alternatively consists of recombinantproteins, or fragments thereof, of RNA-phage AP205.

The AP205 genome consists of a maturation protein, a coat protein, areplicase and two open reading frames not present in related phages; alysis gene and an open reading frame playing a role in the translationof the maturation gene (Klovins, J., et al., J. Gen. Virol. 83: 1523-33(2002)). AP205 coat protein can be expressed from plasmid pAP283-58 (SEQID NO: 11), which is a derivative of pQb10 (Kozlovska, T. M. et al.,Gene 137:133-37 (1993)), and which contains an AP205 ribosomal bindingsite. Alternatively, AP205 coat protein may be cloned into pQb185,downstream of the ribosomal binding site present in the vector. Bothapproaches lead to expression of the protein and formation of capsids asdescribed in the co-pending U.S. Provisional Appl. No. 60/396,126, filedJul. 17, 2002, which is incorporated by reference in its entirety, andin particular as described in Example 2 of said patent application.Vectors pQb10 and pQb185 are vectors derived from pGEM vector, andexpression of the cloned genes in these vectors is controlled by the trppromoter (Kozlovska, T. M. et al., Gene 137:133-37 (1993)). PlasmidpAP283-58 (SEQ ID NO:11) comprises a putative AP205 ribosomal bindingsite in the following sequence, which is downstream of the XbaI site,and immediately upstream of the ATG start codon of the AP205 coatprotein: tctagaATTTTCTGCGCACCCAT CCCGGGTGGCGCCCAAA-GTGAGGAAAATCACatg.The vector pQb185 comprises a Shine Delagarno sequence downstream fromthe XbaI site and upstream of the start codon (tctagaTTAACCCAACGCGTAGGAGTCAGGCCatg, Shine Delagarno sequence underlined).

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of recombinant coat proteins, or fragmentsthereof, of the RNA-phage AP205.

This preferred embodiment of the present invention, thus, comprisesAP205 coat proteins that form capsids. Such proteins are recombinantlyexpressed, or prepared from natural sources. AP205 coat proteinsproduced in bacteria spontaneously form capsids, as evidenced byElectron Microscopy (EM) and immunodiffusion. The structural propertiesof the capsid formed by the AP205 coat protein (SEQ ID NO: 12) and thoseformed by the coat protein of the AP205 RNA phage are nearlyindistinguishable when seen in EM. AP205 VLPs are highly immunogenic,and can be linked with antigens and/or antigenic determinants togenerate vaccine constructs displaying the antigens and/or antigenicdeterminants oriented in a repetitive manner. High titers are elicitedagainst the so displayed antigens showing that bound antigens and/orantigenic determinants are accessible for interacting with antibodymolecules and are immunogenic.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of recombinant mutant coat proteins, orfragments thereof, of the RNA-phage AP205.

Assembly-competent mutant forms of AP205 VLPs, including AP205 coatprotein with the subsitution of proline at amino acid 5 to threonine(SEQ ID NO: 13), may also be used in the practice of the invention andleads to a further preferred embodiment of the invention. These VLPs,AP205 VLPs derived from natural sources, or AP205 viral particles, maybe bound to antigens to produce ordered repetitive arrays of theantigens in accordance with the present invention.

AP205 P5-T mutant coat protein can be expressed from plasmid pAP281-32(SEQ ID No. 14), which is derived directly from pQb185, and whichcontains the mutant AP205 coat protein gene instead of the Qβ coatprotein gene. Vectors for expression of the AP205 coat protein aretransfected into E. coli for expression of the AP205 coat protein.

Methods for expression of the coat protein and the mutant coat protein,respectively, leading to self-assembly into VLPs are described inco-pending U.S. Provisional Appl. No. 60/396,126, filed Jul. 17, 2002,which is incorporated by reference in its entirety. Suitable E. colistrains include, but are not limited to, E. coli K802, JM 109, RR1.Suitable vectors and strains and combinations thereof can be identifiedby testing expression of the coat protein and mutant coat protein,respectively, by SDS-PAGE and capsid formation and assembly byoptionally first purifying the capsids by gel filtration andsubsequently testing them in an immunodiffusion assay (Ouchterlony test)or Electron Microscopy (Kozlovska, T. M. et al., Gene 137:133-37(1993)).

AP205 coat proteins expressed from the vectors pAP283-58 and pAP281-32may be devoid of the initial Methionine amino acid, due to processing inthe cytoplasm of E. coli. Cleaved, uncleaved forms of AP205 VLP, ormixtures thereof are further preferred embodiments of the invention.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of a mixture of recombinant coat proteins, orfragments thereof, of the RNA-phage AP205 and of recombinant mutant coatproteins, or fragments thereof, of the RNA-phage AP205.

In a further preferred embodiment of the present invention, thevirus-like particle comprises, or alternatively essentially consists of,or alternatively consists of fragments of recombinant coat proteins orrecombinant mutant coat proteins of the RNA-phage AP205.

Recombinant AP205 coat protein fragments capable of assembling into aVLP and a capsid, respectively are also useful in the practice of theinvention. These fragments may be generated by deletion, eitherinternally or at the termini of the coat protein and mutant coatprotein, respectively. Insertions in the coat protein and mutant coatprotein sequence or fusions of antigen sequences to the coat protein andmutant coat protein sequence, and compatible with assembly into a VLP,are further embodiments of the invention and lead to chimeric AP205 coatproteins, and particles, respectively. The outcome of insertions,deletions and fusions to the coat protein sequence and whether it iscompatible with assembly into a VLP can be determined by electronmicroscopy.

The particles formed by the AP205 coat protein, coat protein fragmentsand chimeric coat proteins described above, can be isolated in pure formby a combination of fractionation steps by precipitation and ofpurification steps by gel filtration using e.g. Sepharose CL-4B,Sepharose CL-2B, Sepharose CL-6B columns and combinations thereof asdescribed in the co-pending U.S. Provisional Appl. No. 60/396,126, filedJul. 17, 2002, which is incorporated by reference in its entirety. Othermethods of isolating virus-like particles are known in the art, and maybe used to isolate the virus-like particles (VLPs) of bacteriophageAP205. For example, the use of ultracentrifugation to isolate VLPs ofthe yeast retrotransposon Ty is described in U.S. Pat. No. 4,918,166,which is incorporated by reference herein in its entirety.

According to the present invention, one or more angiotensin peptidemoieties may be attached to one subunit of the capsid of RNA phages coatproteins. The ability to couple several angiotensin peptide moieties persubunit of the capsid of the coat protein of RNA phages and inparticular of Qβ capsid allows for the generation of a dense angiotensinpeptide moiety array. Other viral capsids may be used for covalentattachment of angiotensin peptide moieties by way of chemicalcross-linking, such for example a HBcAg modified with a lysine residuein its major immunodominant region (MIR; WO 00/32227). The distancebetween the spikes (corresponding to the MIR) of HBcAg is 50 Angstroms(Wynne, S A. et al., Mol. Cell 3: 771-780 (1999)), and therefore anangiotensin peptide moiety array with distances shorter than 50 A cannotbe generated.

Capsids of Qβ coat protein display a defined number of lysine residueson their surface, with a defined topology with three lysine residuespointing towards the interior of the capsid and interacting with theRNA, and four other lysine residues exposed to the exterior of thecapsid. These defined properties favor the attachment of angiotensinpeptide moieties to the exterior of the particle, and not to theinterior where the lysine residues interact with RNA. Capsids of otherRNA phage coat proteins also have a defined number of lysine residues ontheir surface and a defined topology of these lysine residues. Anotheradvantage of the capsids derived from RNA phages is their highexpression yield in bacteria, that allows the production of largequantities of material at affordable cost.

Another feature of the capsid of Qβ coat protein is its stability. Qβsubunits are bound via disulfide bridges to each other, covalentlylinking the subunits. Qβ capsid protein also shows unusual resistance toorganic solvents and denaturing agents. Surprisingly, we have observedthat DMSO and acetonitrile concentrations as high as about 30%, andGuanidinium concentrations as high as about 1 M could be used withoutaffecting the stability or the ability to form angiotensin peptidemoiety arrays of the capsid. Thus, theses organic solvents may be usedto couple hydrophobic molecules, such as certain angiotensin peptidemoieties. The high stability of the capsid of Qβ coat protein is animportant feature pertaining to its use for immunization and vaccinationof mammals and humans in particular. The resistance of the capsid toorganic solvent allows the coupling of angiotensin peptide moieties orderiviatives thereof that are not soluble in aqueous buffers.

Insertion of a cysteine residue into the N-terminal α-hairpin of thecoat protein of the RNA phage MS-2 has been described in the U.S. Pat.No. 5,698,424, which is incorporated by reference herein in itsentirety. We note however, that the presence of an exposed free cysteineresidue in the capsid may lead to oligomerization of capsids by way ofdisulfide bridge formation. Other attachments contemplated in the aboveU.S. patent involve the formation of disulfide bridges between theangiotensin peptide moieties and the Qβ particle. Such attachments arelabile to sulfhydryl-moiety containing molecules.

The reaction between an initial disulfide bridge formed with acysteine-residue on Qβ, and the antigen containing a free sulfhydrylresidue releases sulfhydryl containing species other than theangiotensin peptide moiety. These newly formed sulfhydryl containingspecies can react again with other disulfide bridges present on theparticle, thus establishing an equilibrium. Upon reaction with thedisulfide bridge formed on the particle, the angiotensin peptide moietymay either form a disulfide bridge with the cysteine-residue from theparticle, or with the cysteine-residue of the leaving group moleculewhich was forming the initial disulfide bridge on the particle.Moreover, the other method of attachment described, using ahetero-bifunctional cross-linker reacting with a cysteine on the Qβparticle on one side, and with a lysine residue on the angiotensinpeptide moiety on the other side, may lead to a random orientation ofthe angiotensin peptide moieties on the particle.

We further note that, in contrast to the capsid of the Qβ and Fr coatproteins, recombinant MS-2 described in U.S. Pat. No. 5,698,424 isessentially free of nucleic acids, while RNA is packaged inside the twocapsids mentioned above.

We describe here new and inventive conjugates and conjugates allowingthe formation of robust arrays of angiotensin peptide moieties, withvariable density of angiotensin epitopes in the conjugates. We show thatvery high epitope density can be achieved by attaching angiotensinpeptide moieties to VLPs. Further, the density and spacing ofangiotensin peptide moieties can be modified by alterations in thenumber and type of residues with suitable first attachment sites. Forexample copending U.S. patent application Ser. No. 10/050,902, filedJan. 18, 2002, discloses a Qβ mutant coat protein with additional lysineresidues, suitable for obtaining higher density arrays than observedwith wild type Qβ coat protein. Further, the aforesaid application alsodiscloses conjugates suitable for simultaneous display of severalantigens with appropriate spacing, and conjugates wherein the additionof accessory molecules, enhancing solubility or modifiying the capsid ina suitable and desired way. Other Qβ coat protein mutants, formingcapsids, which are virus-like particles, are disclosed in copending U.S.patent application Ser. No. 10/050,902, and are suitable for generatingcompositions of the invention. In particular, in occurrences wheresolubility of the angiotensin peptide moiety, and of the Qβ-angiotensinpeptide antigen array imposes a limit on the number of angiotensinpeptide moieties that can be attached on the Qβ virus-like particle,mutants where lysine residues have been substituted for arginines, whichdo not have the same reactivity as lysine residues, can be used. Whenpreparing these compositions, a high concentration of angiotensinpeptide moiety, or angiotensin peptide moiety modified to comprise asecond attachment site, can be used to achieve complete reaction at thelysine residues on the mutant Qβ virus-like particles, withoutgenerating potentially insoluble particles with a higher number ofattached angiotensin peptide moieties, as would be the case when usingthe wt Qβ virus-like particle.

The crystal structure of several RNA bacteriophages has been determined(Golmohammadi, R. et al., Structure 4:543-554 (1996)). Using suchinformation, one skilled in the art could readily identify surfaceexposed residues and modify bacteriophage coat proteins such that one ormore reactive amino acid residues can be inserted. Thus, one skilled inthe art could readily generate and identify modified forms ofbacteriophage coat proteins which can be used in the practice of theinvention. Thus, variants of proteins which form capsids or capsid-likestructures (e.g., coat proteins of bacteriophage Qβ, bacteriophage R17,bacteriophage fr, bacteriophage GA, bacteriophage SP, and bacteriophageMS2) can also be used to prepare vaccine conjugates of the invention.

Although the sequence of the variants proteins discussed above willdiffer from their wild-type counterparts, these variant proteins willgenerally retain the ability to form capsids or capsid-like structures.Thus, the invention further includes vaccine conjugates which containvariants of proteins which form capsids or capsid-like structures, aswell as methods for preparing such vaccine conjugates, individualprotein subunits used to prepare such vaccine conjugates. Thus, includedwithin the scope of the invention are variant forms of wild-typeproteins which form ordered and repetitive arrays (e.g., variants ofproteins which form capsids or capsid-like structures) and retain theability to associate and form capsids or capsid-like structures.Normally, C- an N-terminal trunction variants retain the ability to formvirus like particles. As a result, variant forms including deletion,addition, or subsitution, chimeric forms, and naturally occuringvariants are suitable components of the invention.

Bacterial Pili and pilin proteins. In other embodiments, a bacterialpilin, a subportion of a bacterial pilin, or a fusion protein whichcontains either a bacterial pilin or subportion thereof is used toprepare vaccine conjugates of the invention. Examples of pilin proteinsinclude pilins produced by Escherichia coli, Haemophilus influenzae,Neisseria meningitidis, Neisseria gonorrhoeae, Caulobacter crescentus,Pseudomonas stutzeri, and Pseudomonas aeruginosa. The amino acidsequences of pilin proteins suitable for use with the present inventioninclude those set out in GenBank reports AJ000636, AJ132364, AF229646,AF051814, AF051815, and X00981, the entire disclosures of which areincorporated herein by reference.

Bacterial pilin proteins are generally processed to remove N-terminalleader sequences prior to export of the proteins into the bacterialperiplasm. Further, as one skilled in the art would recognize, bacterialpilin proteins used to prepare vaccine conjugates of the invention willgenerally not have the naturally present leader sequence.

One specific example of a pilin protein suitable for use in the presentinvention is the P-pilin of E. coli (GenBank report AF237482). Anexample of a Type-1 E. coli pilin suitable for use with the invention isa pilin having the amino acid sequence set out in GenBank report P04128(SEQ ID NO:2), which is encoded by nucleic acid having the nucleotidesequence set out in GenBank report M27603. The entire disclosures ofthese GenBank reports are incorporated herein by reference. Again, themature form of the above referenced protein would generally be used toprepare vaccine conjugates of the invention.

Bacterial pilins or pilin subportions suitable for use in the practiceof the present invention will generally be able to associate to formnon-natural molecular scaffolds. Methods for preparing pili andpilus-like structures in vitro are known in the art. Bullitt et al.,Proc. Natl. Acad. Sci. USA 93:12890-12895 (1996), for example, describethe in vitro reconstitution of E. coli P-pili subunits. Further, Eshdatet al (J. Bacteriol. 148:308-314 (1981)) describe methods suitable fordissociating Type-1 pili of E. coli and the reconstitution of pili. Inbrief, these methods are as follows: pili are dissociated by incubationat 37° C. in saturated guanidine hydrochloride. Pilin proteins are thenpurified by chromatography, after which pilin dimers are formed bydialysis against 5 mM tris(hydroxymethyl) aminomethane hydrochloride (pH8.0). Eshdat et al. also found that pilin dimers reassemble to form piliupon dialysis against the 5 mM tris(hydroxymethyl) aminomethane (pH 8.0)containing 5 mM MgCl₂.

Further, using, for example, conventional genetic engineering andprotein modification methods, pilin proteins may be modified to containa first attachment site to which an angiotensin peptide moiety is linkedthrough a second attachment site. Alternatively, angiotensin peptidemoieties can be directly linked through a second attachment site toamino acid residues which are naturally resident in these proteins.These modified pilin proteins may then be used in immunizing conjugatesof the invention.

Bacterial pilin proteins used to prepare conjugates of the invention maybe modified in a manner similar to that described herein for HBcAg. Forexample, cysteine and lysine residues may be either deleted orsubstituted with other amino acid residues and first attachment sitesmay be added to these proteins. Further, pilin proteins may either beexpressed in modified form or may be chemically modified afterexpression. Similarly, intact pili may be harvested from bacteria andthen modified chemically.

In another embodiment, pili or pilus-like structures are harvested frombacteria (e.g., E. coli) and used to form vaccine conjugates of theinvention. One example of pili suitable for preparing vaccine conjugatesis the Type-1 pilus of E. coli, which is formed from pilin monomershaving the amino acid sequence set out in SEQ ID NO:2.

A number of methods for harvesting bacterial pili are known in the art.Bullitt and Makowski (Biophys. J. 74:623-632 (1998)), for example,describe a pilus purification method for harvesting P-pili from E. coli.According to this method, pili are sheared from hyperpiliated E. colicontaining a P-pilus plasmid and purified by cycles of solubilizationand MgCl₂ (1.0 M) precipitation. Copending U.S. patent application Ser.No. 10/050,902, filed Jan. 18, 2002, discloses harvesting andpurification of Type I pili from bacteria that naturally produce pili,or into which a vector has been introduced encoding the fim operonresponsible for pilus production.

Once harvested, pili or pilus-like structures may be modified in avariety of ways. For example, a first attachment site can be added tothe pili to which one or more angiotensin peptide moieties may beattached through a second attachment site. In other words, bacterial pilor pilus-like structures can be harvested and modified to formnon-natural molecular scaffolds.

Pili or pilus-like structures may also be modified by the attachment ofangiotensin peptide moieties in the absence of a non-natural firstattachment site. For example, antigens or antigenic determinants couldbe linked to naturally occurring cysteine resides or lysine residues. Insuch instances, the high order and repetitiveness of a naturallyoccurring amino acid residue would guide the coupling of the angiotensinpeptide moieties to the pili or pilus-like structures. For example, thepili or pilus-like structures could be linked to the second attachmentsites of the angiotensin peptide moieties using a heterobifunctionalcross-linking agent.

When structures which are naturally synthesized by organisms (e.g.,pili) are used to prepare vaccine conjugates of the invention, it willoften be advantageous to genetically engineer these organisms so thatthey produce structures having desirable characteristics. For example,when Type-1 pili of E. coli are used, the E. coli from which these piliare harvested may be modified so as to produce structures with specificcharacteristics. Examples of possible modifications of pilin proteinsinclude the insertion of one or more lysine residues, the deletion orsubstitution of one or more of the naturally resident lysine residues,and the deletion or substitution of one or more naturally residentcysteine residues (e.g., the cysteine residues at positions 44 and 84 inSEQ ID NO:2).

Further, additional modifications can be made to pilin genes whichresult in the expression products containing a first attachment siteother than a lysine residue (e.g., a FOS or JUN domain). Of course,suitable first attachment sites will generally be limited to those whichdo not prevent pilin proteins from forming pili or pilus-like structuressuitable for use in vaccine conjugates of the invention. The ability ofrecombinant pilin proteins to form pili may be determined by a number ofmethods including electron microscopy.

Pilin genes which naturally reside in bacterial cells can be modified invivo (e.g., by homologous recombination) or pilin genes with particularcharacteristics can be inserted into these cells. For examples, pilingenes could be introduced into bacterial cells as a component of eithera replicable cloning vector or a vector which inserts into the bacterialchromosome. The inserted pilin genes may also be linked to expressionregulatory control sequences (e.g., a lac operator).

In most instances, the pili or pilus-like structures used in vaccineconjugates of the invention will be composed of single type of a pilinsubunit. However, the conjugates of the invention also include vaccinescomprising pili or pilus-like structures formed from heterogenous pilinsubunits. Pili or pilus-like structures composed of identical subunitswill generally be used because they are expected to form structureswhich present highly ordered and repetitive antigen arrays.

Second attachment site. The preparation of molecular scaffolds withordered and repetitive arrays is provided by the present includingconjugates of capsids of RNA phage coat proteins with a high epitopedensity. The nature of the angiotensin peptide moiety, and nature andlocation of the second attachment site on the moiety are importantfactors that may influence the means available to construct conjugatesof the invention, and the effectiveness of those conjugates in inducingan immune response, as is understood by those of ordinary skill in theart.

A prerequisite for designing a second attachment site is the choice ofthe position at which it should be fused, inserted or generallyengineered or attached. A skilled artisan would know how to findguidance in selecting the position of the second attachment site, andmany factors may be considered relevant to this decision. The chemicaland/or crystal structure of the angiotensin peptide moiety may provideinformation on the availability of domains on the molecule suitable forcoupling. A reactive domain's accessibility to solvent may be a limitingfactor in the kinetics of chemical coupling to a first attachment site.Groups suitable for coupling must be available, such as sulfhydrylresidues. In general, in the case where immunization with an angiotensinpeptide moiety is aimed at inhibiting the interaction of the angiotensinpeptide moiety, which may also be a self-antigen with its naturalligands, such as a substrate or a receptor, the second attachment sitewill be added such that it allows generation of antibodies against thesite of interaction with the natural ligands. Thus, the location of thesecond attachment site will selected such, that steric hindrance fromthe second attachment site or any amino acid linker containing it, isavoided. In further embodiments, an antibody response directed at a sitedistinct from the interaction site of the antigen with its naturalligand is desired. In such embodiments, the second attachment site maybe selected such that it prevents generation of antibodies against theinteraction site of the antigen with its natural ligands. Other factorsof consideration include the nature of the angiotensin peptide moiety,its biochemical properties, such as pI, charge distribution, furthermodification. In general, flexible linkers are favored.

Other criteria in selecting the position of the second attachment siteinclude the oligomerization state of the angiotensin peptide moiety, thesite of oligomerization, the presence of a cofactor, and theavailability of experimental evidence disclosing sites in the moietystructure and sequence where modification of the moiety is compatiblewith the function moiety, or with the generation of antibodiesrecognizing the moiety and preferably, blocking function of theangiotensin peptide moiety. In certain embodiments, one or moreadditional amino acids (leading to a non-naturally occurring secondattachment site) are added either at the C- or at the N-terminus of theangiotensin peptide moiety sequences in order to assure, in particular,an oriented and ordered association of the angiotensin peptide moiety tothe virus-like particle in accordance with the present invention.

A particularly favored method of attachment of polypeptide antigens toVLPs, and in particular to capsids of RNA phage coat proteins, is thelinking of a lysine residue on the surface of the capsid of RNA phagecoat proteins with a sulfhydryl group residue on the antigen, such as isfound in cysteine residues. Similarly, free sulfhydryl groups onangiotensin peptide moieties may also be effective attachment sites.Where an oxidized sulfhydryl groups must be in a reduced state in orderto function as a second attachment site, reduction may be achieved withe.g. DTT, TCEP or β-mercaptoethanol.

According to the present invention, the epitope density on the capsid ofRNA phage coat proteins can be modulated by the choice of cross-linkerand other reaction conditions. For example, the cross-linkers Sulfo-GMBSand SMPH allow reaching high epitope density. Derivatization ispositively influenced by high concentration of reactants, andmanipulation of the reaction conditions can be used to control thenumber of antigens coupled to RNA phages capsid proteins, and inparticular to Qβ capsid protein. In addition, the number of firstattachment sites on the core particle is another factor affecting thedensity of the angiotensin peptide moiety array. In one embodiment ofthe present invention, we provide a Qβ mutant coat protein withadditional lysine residues, suitable for obtaining higher densityarrays.

In the most preferred embodiments, the angiotensin peptide moietycomprises a single second attachment site or a single reactiveattachment site capable of association with the first attachment siteson the core particle and the VLPs or VLP subunits, respectively. Thisensures a defined and uniform binding and association, respectively, ofthe at least one, but typically more than one, preferably more than 10,20, 40, 80, 120 antigens to the core particle and VLP, respectively. Theprovision of a single second attachment site or a single reactiveattachment site on the antigen, thus, ensures a single and uniform typeof binding and association, respectively leading to a very highlyordered and repetitive array. For example, if the binding andassociation, respectively, is effected by way of a lysine—(as the firstattachment site) and cysteine—(as a second attachment site) interaction,it is ensured, in accordance with this preferred embodiment of theinvention, that only one cysteine residue per antigen, independentwhether this cysteine residue is naturally or non-naturally present onthe antigen, is capable of binding and associating, respectively, withthe VLP and the first attachment site of the core particle,respectively.

In a further preferred embodiment of the invention, the covalent is anon-peptide bond.

In some embodiments, engineering of a second attachment site onto theantigen require the fusion of an amino acid linker containing an aminoacid suitable as second attachment site according to the disclosures ofthis invention. Therefore, in a preferred embodiment of the presentinvention, an amino acid linker is bound to the antigen or the antigenicdeterminant by way of at least one covalent bond. Preferably, the aminoacid linker comprises, or alternatively consists of, the secondattachment site. In a further preferred embodiment, the amino acidlinker comprises a sulfhydryl group or a cysteine residue. In anotherpreferred embodiment, the amino acid linker is cysteine. Some criteriaof selection of the amino acid linker as well as further preferredembodiments of the amino acid linker according to the invention havealready mentioned above.

In a further preferred embodiment of the invention, the at least oneantigen or antigenic determinant, i.e. the PrP protein, PrP peptide orthe PrP domain is fused to the core particle and the virus-likeparticle, respectively. As outlined above, a VLP is typically composedof at least one subunit assembling into a VLP. Thus, in again a furtherpreferred embodiment of the invention, the antigen or antigenicdeterminant, preferably the at least one angiotensin peptide moiety, isfused to at least one subunit of the virus-like particle or of a proteincapable of being incorporated into a VLP generating a chimericVLP-subunit-angiotensin peptide moiety fusion.

Fusion of the angiotensin peptide moieties can be effected by insertioninto the VLP subunit sequence, or by fusion to either the N- orC-terminus of the VLP-subunit or protein capable of being incorporatedinto a VLP. Hereinafter, when referring to fusion proteins of a peptideto a VLP subunit, the fusion to either ends of the subunit sequence orinternal insertion of the peptide within the subunit sequence areencompassed.

Fusion may also be effected by inserting the angiotensin peptide moietysequences into a variant of a VLP subunit where part of the subunitsequence has been deleted, that are further referred to as truncationmutants. Truncation mutants may have N- or C-terminal, or internaldeletions of part of the sequence of the VLP subunit. For example, thespecific VLP HBcAg with, for example, deletion of amino acid residues 79to 81 is a truncation mutant with an internal deletion. Fusion ofangiotensin peptide moieties to either the N- or C-terminus of thetruncation mutants VLP-subunits also lead to embodiments of theinvention. Likewise, fusion of an epitope into the sequence of the VLPsubunit may also be effected by substitution, where for example for thespecific VLP HBcAg, amino acids 79-81 are replaced with a foreignepitope. Thus, fusion, as referred to hereinafter, may be effected byinsertion of the angiotensin peptide moiety sequence in the sequence ofa VLP subunit, by substitution of part of the sequence of the VLPsubunit with the angiotensin peptide moiety sequence, or by acombination of deletion, substitution or insertions.

The chimeric angiotensin peptide moiety-VLP subunit will be in generalcapable of self-assembly into a VLP. VLP displaying epitopes fused totheir subunits are also herein referred to as chimeric VLPs. Asindicated, the virus-like particle comprises or alternatively iscomposed of at least one VLP subunit. In a further embodiment of theinvention, the virus-like particle comprises or alternatively iscomposed of a mixture of chimeric VLP subunits and non-chimeric VLPsubunits, i.e. VLP subunits not having an antigen fused thereto, leadingto so called mosaic particles. This may be advantageous to ensureformation of and assembly to a VLP. In those embodiments, the proportionof chimeric VLP-subunits may be 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80,90, 95% or higher.

Flanking amino acid residues may be added to either end of the sequenceof the peptide or epitope to be fused to either end of the sequence ofthe subunit of a VLP, or for internal insertion of such peptidicsequence into the sequence of the subunit of a VLP. Glycine and serineresidues are particularly favored amino acids to be used in the flankingsequences added to the angiotensin peptide moiety to be fused. Glycineresidues confer additional flexibility, which may diminish thepotentially destabilizing effect of fusing a foreign sequence into thethe sequence of a VLP subunit.

In a specific embodiment of the invention, the VLP is a Hepatitis B coreantigen VLP. Fusion proteins to either the N-terminus of a HBcAg(Neyrinck, S. et al., Nature Med. 5:1157-1163 (1999)) or insertions inthe so called major immunodominant region (MIR) have been described(Pumpens, P. and Grens, E., Intervirology 44:98-114 (2001)), WO01/98333), and are preferred embodiments of the invention. Naturallyoccurring variants of HBcAg with deletions in the MIR have also beendescribed (Pumpens, P. and Grens, E., Intervirology 44:98-114 (2001),which is expressly incorporated by reference in their entirety), andfusions to the N- or C-terminus, as well as insertions at the positionof the MIR corresponding to the site of deletion as compared to a wtHBcAg are further embodiments of the invention. Fusions to theC-terminus have also been described (Pumpens, P. and Grens, E.,Intervirology 44:98-114 (2001)). One skilled in the art will easily findguidance on how to construct fusion proteins using classical molecularbiology techniques (Sambrook, J. et al., eds., Molecular Cloning, ALaboratory Manual, 2nd. edition, Cold Spring Habor Laboratory Press,Cold Spring Harbor, N.Y. (1989), Ho et al., Gene 77:51 (1989)). Vectorsand plasmids encoding HBcAg and HBcAg fusion proteins and useful for theexpression of a HBcAg and HBcAg fusion proteins have been described(Pumpens, P. & Grens, E. Intervirology 44: 98-114 (2001), Neyrinck, S.et al., Nature Med. 5:1157-1163 (1999)) and can be used in the practiceof the invention. We also describe by way of example (Example 6) theinsertion of an epitope into the MIR of HBcAg, resulting in a chimericself-assembling HBcAg. An important factor for the optimization of theefficiency of self-assembly and of the display of the epitope to beinserted in the MIR of HBcAg is the choice of the insertion site, aswell as the number of amino acids to be deleted from the HBcAg sequencewithin the MIR (Pumpens, P. and Grens, E., Intervirology 44:98-114(2001); EP 0 421 635; U.S. Pat. No. 6,231,864) upon insertion, or inother words, which amino acids form HBcAg are to be substituted with thenew epitope. For example, substitution of HBcAg amino acids 76-80,79-81, 79-80, 75-85 or 80-81 with foreign epitopes has been described(Pumpens, P. and Grens, E., Intervirology 44:98-114 (2001); EP 0 421635; U.S. Pat. No. 6,231,864). HBcAg contains a long arginine tail(Pumpens, P. and Grens, E., Intervirology 44:98-114 (2001)) which isdispensable for capsid assembly and capable of binding nucleic acids(Pumpens, P. and Grens, E., Intervirology 44:98-114 (2001)). HBcAgeither comprising or lacking this arginine tail are both embodiments ofthe invention.

In a further preferred embodiment of the invention, the VLP is a VLP ofa RNA phage. The major coat proteins of RNA phages spontaneouslyassemble into VLPs upon expression in bacteria, and in particular in E.coli. Specific examples of bacteriophage coat proteins which can be usedto prepare compositions of the invention include the coat proteins ofRNA bacteriophages such as bacteriophage Qβ (SEQ ID NO:10; PIR Database,Accession No. VCBPQβ referring to Qβ CP and SEQ ID NO: 11; Accession No.AAA16663 referring to Qβ A1 protein) and bacteriophage fr (SEQ ID NO:4;PIR Accession No. VCBPFR).

In a more preferred embodiment, the at least one angiotensin peptidemoiety is fused to a Qβ coat protein. Fusion protein constructs whereinepitopes have been fused to the C-terminus of a truncated form of the A1protein of Qβ, or inserted within the A1 protein have been described(Kozlovska, T. M., et al., Intervirology, 39:9-15 (1996)). The A1protein is generated by suppression at the UGA stop codon and has alength of 329 aa, or 328 aa, if the cleavage of the N-terminalmethionine is taken into account. Cleavage of the N-terminal methioninebefore an alanine (the second amino acid encoded by the Qβ CP gene)usually takes place in E. coli, and such is the case for N-termini ofthe Qβ coat proteins CP. The part of the A1 gene, 3′ of the UGA ambercodon encodes the CP extension, which has a length of 195 amino acids.Insertion of the at least one angiotensin peptide moiety betweenposition 72 and 73 of the CP extension leads to further embodiments ofthe invention (Kozlovska, T. M., et al., Intervirology 39:9-15 (1996)).Fusion of a angiotensin peptide moiety at the C-terminus of aC-terminally truncated Qβ A1 protein leads to further preferredembodiments of the invention. For example, Kozlovska et al.,(Intervirology, 39: 9-15 (1996)) describe Qβ A1 protein fusions wherethe epitope is fused at the C-terminus of the Qβ CP extension truncatedat position 19.

As described by Kozlovska et al. (Intervirology, 39: 9-15 (1996)),assembly of the particles displaying the fused epitopes typicallyrequires the presence of both the A1 protein-angiotensin peptide moietyfusion and the wt CP to form a mosaic particle. However, embodimentscomprising virus-like particles, and hereby in particular the VLPs ofthe RNA phage Qβ coat protein, which are exclusively composed of VLPsubunits having at least one angiotensin peptide moiety fused thereto,are also within the scope of the present invention.

The production of mosaic particles may be effected in a number of ways.Kozlovska et al., Intervirolog, 39:9-15 (1996), describe two methods,which both can be used in the practice of the invention. In the firstapproach, efficient display of the fused epitope on the VLPs is mediatedby the expression of the plasmid encoding the Qβ A1 protein fusionhaving a UGA stop codong between CP and CP extension in a E. coli strainharboring a plasmid encoding a cloned UGA suppressor tRNA which leads totranslation of the UGA codon into Trp (pISM3001 plasmid (Smiley B. K.,et al., Gene 134:33-40 (1993))). In another approach, the CP gene stopcodon is modified into UAA, and a second plasmid expressing the A1protein-angiotensin peptide moiety fusion is cotransformed. The secondplasmid encodes a different antibiotic resistance and the origin ofreplication is compatible with the first plasmid (Kozlovska, T. M., etal., Intervirology 39:9-15 (1996)). In a third approach, CP and the A1protein-angiotensin peptide moiety fusion are encoded in a bicistronicmanner, operatively linked to a promoter such as the Trp promoter, asdescribed in FIG. 1 of Kozlovska et al., Intervirology, 39:9-15 (1996).

In a further embodiment, the angiotensin peptide moiety is insertedbetween amino acid 2 and 3 (numbering of the cleaved CP, that is whereinthe N-terminal methionine is cleaved) of the fr CP, thus leading to aangiotensin peptide moiety −fr CP fusion protein. Vectors and expressionsystems for construction and expression of fr CP fusion proteinsself-assembling to VLP and useful in the practice of the invention havebeen described (Pushko P. et al., Prot. Eng. 6:883-891 (1993)). In aspecific embodiment, the angiotensin peptide moiety sequence is insertedinto a deletion variant of the fr CP after amino acid 2, whereinresidues 3 and 4 of the fr CP have been deleted (Pushko P. et al., Prot.Eng. 6:883-891 (1993)).

Fusion of epitopes in the N-terminal protuberant β-hairpin of the coatprotein of RNA phage MS-2 and subsequent presentation of the fusedepitope on the self-assembled VLP of RNA phage MS-2 has also beendescribed (WO 92/13081), and fusion of angiotensin peptide moiety byinsertion or substitution into the coat protein of MS-2 RNA phage isalso falling under the scope of the invention.

In another embodiment of the invention, the angiotensin peptide moietiesare fused to a capsid protein of papillomavirus. In a more specificembodiment, the angiotensin peptide moieties are fused to the majorcapsid protein L1 of bovine papillomavirus type I (BPV-1). Vectors andexpression systems for construction and expression of BPV-1 fusionproteins in a baculovirus/insect cells systems have been described(Chackerian, B. et al., Proc. Natl. Acad. Sci. USA 96:2373-2378 (1999),WO 00/23955). Substitution of amino acids 130-136 of BPV-1 L1 with aangiotensin peptide moiety leads to a BPV-1 L1-angiotensin peptidemoiety fusion protein, which is a preferred embodiment of the invention.Cloning in a baculovirus vector and expression in baculovirus infectedSf9 cells has been described, and can be used in the practice of theinvention (Chackerian, B. et al., Proc. Natl. Acad. Sci. USA96:2373-2378 (1999), WO 00/23955). Purification of the assembledparticles displaying the fused angiotensin peptide moieties can beperformed in a number of ways, such as for example gel filtration orsucrose gradient ultracentrifugation (Chackerian, B. et al., Proc. Natl.Acad. Sci. USA 96:2373-2378 (1999), WO 00/23955).

In a further embodiment of the invention, the angiotensin peptidemoieties are fused to a Ty protein capable of being incorporated into aTy VLP. In a more specific embodiment, the angiotensin peptide moietiesare fused to the p1 or capsid protein encoded by the TYA gene (Roth, J.F., Yeast 16:785-795 (2000)). The yeast retrotransposons Ty1, 2, 3 and 4have been isolated from Saccharomyces Serevisiae, while theretrotransposon Tf1 has been isolated from Schizosaccharomyces Pombae(Boeke, J. D. and Sandmeyer, S. B., “Yeast Transposable Elements,” inThe Molecular and Cellular Biology of the Yeast Saccharomyces: GenomeDynamics, Protein Synthesis, and Energetics., p. 193, Cold Spring HarborLaboratory Press (1991)). The retrotransposons Ty1 and 2 are related tothe copia class of plant and animal elements, while Ty3 belongs to thegypsy family of retrotransposons, which is related to plants and animalretroviruses. In the Ty1 retrotransposon, the p1 protein, also referredto as Gag or capsid protein, has a length of 440 amino acids. P1 iscleaved during maturation of the VLP at position 408, leading to the p2protein, the essential component of the VLP.

Fusion proteins to p1 and vectors for the expression of said fusionproteins in Yeast have been described (Adams, S. E., et al., Nature329:68-70 (1987)). So, for example, a angiotensin peptide moiety may befused to p1 by inserting a sequence coding for the angiotensin peptidemoiety into the BamH1 site of the pMA5620 plasmid (Adams, S. E., et al.,Nature 329:68-70 (1987)). The cloning of sequences coding for foreignepitopes into the pMA5620 vector leads to expression of fusion proteinscomprising amino acids 1-381 of p1 of Ty1-15, fused C-terminally to theN-terminus of the foreign epitope. Likewise, N-terminal fusion ofangiotensin peptide moieties, or internal insertion into the p1sequence, or substitution of part of the p1 sequence is also meant tofall within the scope of the invention. In particular, insertion ofangiotensin peptide moieties into the Ty sequence between amino acids30-31, 67-68, 113-114 and 132-133 of the Ty protein p1 (EP0677111) leadsto preferred embodiments of the invention.

Further VLPs suitable for fusion of angiotensin peptide moieties are,for example, Retrovirus-like-particles (WO9630523), HIV2 Gag (Kang, Y.C., et al, Biol. Chem. 380:353-364 (1999)), Cowpea Mosaic Virus (Taylor,K. M. et al., Biol. Chem. 380:387-392 (1999)), parvovirus VP2 VLP(Rueda, P. et al., Virology 263:89-99 (1999)), HBsAg (U.S. Pat. No.4,722,840; EP 0 020 416 B1).

Examples of chimeric VLPs suitable for the practice of the invention arealso those described in Intervirology 39:1 (1996). Further examples ofVLPs contemplated for use in the invention are: HPV-1, HPV-6, HPV-11,HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPV, HIV GAG, Tobacco MosaicVirus. Virus-like particles of SV-40, Polyomavirus, Adenovirus, HerpesSimplex Virus, Rotavirus and Norwalk virus have also been made, andchimeric VLPs of those VLPs are also within the scope of the presentinvention.

Cross linking. Methods for linking the angiotensin peptide moiety to thecore particle are well within the working knowledge of the practitionerof ordinary skill in the art, and numerous references exist to aid sucha practitioner (e.g., Sambrook, J. et al., eds., MOLECULAR CLONING, ALABORATORY MANUAL, 2^(nd). edition, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al., eds., CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc. (1997);Celis, J., ed., CELL BIOLGY, Academic Press, 2^(nd) edition, (1998);Harlow, E. and Lane, D., “Antibodies: A Laboratory Manual,” Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1988), all of which areincorporated herein by reference in their entirities.

Differing methods of achieving an association between the core particleand angiotensin peptide moieity are described herein and are furtherdescribed in copending U.S. patent application Ser. No. 10/050,902,filed Jan. 18, 2002, which is incorporated by reference herein in itsentirety. Methods include the JUN and FOS leucine zipper protein domainsare utilized for the first and second attachment sites of the invention,respectively.

Preferred embodiments of the invention comprise the coupling of thenon-natural molecular scaffold to the angiotensin peptide moiety bychemical cross-linking. There is a wide range of compounds which havebeen developed to facilitate cross-linking of proteins/peptides orconjugation of proteins to derivatized molecules, e.g., angiotensinpeptide moieties. These include, but are not limited, to carboxylic acidderived active esters (activated compounds), mixed anhydrides, acylhalides, acyl azides, alkyl halides, N-maleimides, imino esters,isocyanates and isothiocyanates, which are known to those skilled in theart. These are capable of forming a covalent bond with a reactive groupof a protein molecule. Depending upon the activating group, the reactivegroup is the amino group of a lysine residue on a protein molecule or athiol group in a carrier protein or a modified carrier protein moleculewhich, when reacted, result in amide, amine, thioether, amidine urea orthiourea bond formation. One skilled in the art may identify furthersuitable activating groups, for example, in general reference texts suchas Chemistry of Protein Conjugation and Cross-Linking (Wong (1991) CRCPress, Inc., Boca Raton, Fla.). Most reagents react preferentially withlysine side chain groups.

In some embodiments, the angiotensin peptide moiety is attached to thecore particle by way of chemical cross-linking, using aheterobifunctional cross-linker. Several hetero-bifunctionalcross-linkers are known in the art. In one embodiment, thehetero-bifunctional cross-linker contains a functional group which canreact with the side-chain amino group of lysine residues of the coreparticle, and a functional group which can react with a cysteine residueor sulfhydryl group present, made available for reaction by reduction,or engineered on the angiotensin peptide moiety and optionally also madeavailable for reaction by reduction. The first step of the procedure,called the derivatization, is the reaction of the core particle with thecross-linker. The product of this reaction is an activated coreparticle, also called activated carrier. In the second step, unreactedcross-linker is removed using usual methods such as gel filtration ordialysis. In the third step, the antigen (e.g., the angiotensin peptidemoiety) is reacted with the activated core particle, and this step iscalled the coupling step. Unreacted antigen may be optionally removed ina fourth step.

In an alternative embodiment, the angiotensin peptide moiety isderivatized with an active moiety suitable for cross linking to thefirst attachment site, generating an activated angiotensin peptidemoiety. Such derivatization may occur on an isolated angiotensin peptidemoiety or via a chemical synthesis. The activated angiotensin peptidemoiety is then reacted with the core particle such that coupling occurs.

Several hetero-bifunctional cross-linkers are known in the art. Theseinclude the cross-linkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS,Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and othercross-linkers available, for example from the Pierce Chemical Company(Rockford, Ill., USA), and having one functional group reactive towardsamino groups and one functional group reactive towards SH residues. Theabove mentioned cross-linkers all lead to formation of a thioetherlinkage. Another class of cross-linkers suitable in the practice of theinvention is characterized by the introduction of a disulfide linkagebetween the angiotensin peptide moiety and the core particle uponcoupling. Cross-linkers belonging to this class include for example SPDPand Sulfo-LC-SPDP (Pierce). The extent of derivatization of the coreparticle with cross-linker can be influenced by varying experimentalconditions such as the concentration of each of the reaction partners,the excess of one reagent over the other, the pH, the temperature andthe ionic strength, as is well known from reaction theory in the fieldof organic chemistry. The degree of coupling, i.e. the amount ofangiotensin peptide moiety per carrier can be adjusted by varying theexperimental conditions described above to match the requirements of thevaccine. Solubility of the angiotensin peptide moiety may impose alimitation on the amount of antigen that can be coupled on each subunit,and in those cases where the obtained vaccine is insoluble, reducing theamount of antigens per subunit is beneficial.

In one specific embodiment the chemical agent is the heterobifunctionalcross-linking agent εε-maleimidocaproic acid N-hydroxysuccinimide ester(Tanimori et al., J. Pharm. Dyn. 4:812 (1981); Fujiwara et al., J.Immunol. Meth. 45:195 (1981)), which contains (1) a succinimide groupreactive with amino groups and (2) a maleimide group reactive with SHgroups. A heterologous protein or polypeptide of the first attachmentsite may be engineered to contain one or more lysine residues that willserve as a reactive moiety for the succinimide portion of theheterobifunctional cross-linking agent. Once chemically coupled to thelysine residues of the heterologous protein, the maleimide group of theheterobifunctional cross-linking agent will be available to react withthe SH group of a cysteine residue on the antigen or antigenicdeterminant. Antigen or antigenic determinant preparation in thisinstance may require the engineering of a sulfhydryl residue as thesecond attachment site so that it may be reacted to the free maleimidefunction on the cross-linking agent bound to the non-natural molecularscaffold first attachment sites. Thus, in such an instance, theheterobifunctional cross-linking agent binds to a first attachment siteof the non-natural molecular scaffold and connects the scaffold to asecond binding site of the angiotensin peptide moiety.

Other methods of coupling the angiotensin peptide moiety to the coreparticle include methods wherein the angiotensin peptide moiety iscross-linked to the core particle using carbodiimide bonds. Theseinclude the carbodiimide EDC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride), and NHS.In one method, EDC is mixed with an angiotensin peptide moietycontaining a free carboxylic acid, amino or amido moiety, then added tothe protein carrier. In other methods, the moiety is attached to thecore particle using a homo-bifunctional cross-linker such asglutaraldehyde, DSG, BM[PEO]₄, BS³, (Pierce Chemical Company, Rockford,Ill., USA) or other known homo-bifunctional cross-linkers withfunctional groups reactive towards amine groups or carboxyl groups ofthe core particle.

Additional cross-linking methods and cross-linkers, suitable forattaching a hapten to a core particle and a virus-like particle,respectively, as well as guidance on performing the coupling reactionsand on the use of chemical cross-linkers and chemical cross-linkingprocedures can be found in Hermanson, G. T. in Bioconjugate Techniques,Academic Press Inc., San Diego, Calif., USA.

Further methods of binding the core particle to an angiotensin peptidemoiety include methods where the core particle is biotinylated, and themoiety fused to streptavidin, or methods wherein both the moiety and thecore particle are biotinylated. In this case, the angiotensin peptidemoiety may be first bound to streptavidin or avidin by adjusting theratio of moiety to streptavidin such that free binding sites are stillavailable for binding of the core particle, which is added in the nextstep. Alternatively, all components may be mixed in a “one pot”reaction. Other ligand-receptor pairs, where a soluble form of thereceptor and of the ligand is available, and are capable of beingcross-linked to the core particle or the angiotensin peptide moiety, maybe used as binding agents for binding the angiotensin peptide moiety tothe core particle.

Angiotensin Peptide Moieties

Thus, in one aspect, the invention provides ordered, repetitive arraysof angiotensin peptide moieties suitable for immunization against suchmoieties. Preferred angiotensin peptide moieties are those comprising,or alternatively consisting of, the sequence, or fragments thereof, ofangiotensinogen, angiotensin I or angiotensin II. As noted above, one ormore additional amino acids may be suitably added to either the C- orthe N-terminus of the angiotensin peptide moiety sequences in order toassure, in particular, an oriented and ordered association of theangiotensin peptide moiety to the core particle.

Preferred angiotensin peptide moieties for use in the conjugates andconjugates of the invention are those comprising, or alternativelyconsisting of the full-length sequence of angiotensinogen, angiotensin Ior angiotensin II. Preferably, the angiotensin peptide moietiescomprise, or alternatively consist of, the full-length sequence ofangiotensin II such as CGGDRVYIHPF (referred to herein as “Angio 1”;amino acids in addition to the angiotensin peptide sequence areindicated by italics), or the full-length sequence of angiotensin I,such as CGGDRVYIHPFHL (“Ango 2”), DRVYIHPFHLGGC (“Angio 3”), andCDRVYIHPFHL (“Angio 4”). Further preferred embodiments are thoseangiotensin peptide moieties which comprise, or alternatively consistof, only a fragment of the sequences of angiotensinogen, angiotensin Ior angiotensin II. Certain such embodiments include angiotensin peptidemoieties which comprise, or alternatively consist of, at least threeamino acids of the C-terminus of the angiotensin peptides and, in analternative embodiment, from which at least four amino acids of theN-terminus have been deleted. Other related embodiments are thosederived from angiotensin I such as CHPFHL (“Angio 5”) and CGPFHL (“Angio6”), or those derived from angiotensin II such as CYIHPF (“Angio 7”),CGIHPF (“Angio 8”) and CGGHPF (“Angio 9”).

Additional embodiments of the present invention use angiotensin peptidemoieties which comprise, or alternatively consist of, at least threeamino acids of the N-terminus of the angiotensin peptides and, forwhich, in a further preferred embodiment, at least four, preferablyfive, amino acids of the C-terminus have been deleted. Other relatedembodiments are DRVYIGGC (“Angio 13”), DRVYGGC (“Angio 14”) and DRVGGC(“Angio 15”). It will be understood by those of ordinary skill in theart, however, that the foregoing examples of angiotensin peptidemoieties are non-limiting examples, and that the number and the natureof the amino acids added for coupling, either at C or N terminus, canvary.

In the present invention, it is not necessary that the immunizingangiotensin peptide moiety comprise an entire intact molecule of anyparticular angiotensin peptide moiety. Suitable immune responses againstthe angiotensin peptide moieties of interest may be generated by the useof fragments of the angiotensin peptide moiety, or derivatives, mutantsor muteins thereof.

The invention embodies different sites of linkage and means of linkageof the angiotensin peptide moiety to the core particle, non-limitingexamples of which are described elsewhere herein. Preferred sites andmeans of linkage may also be determined by the ordinarily skilledartisan on the basis of prior experience, theory and by routineexperimentation.

Conjugates, Vaccines and Methods of Use

The invention thus provides conjugates which may be used for preventingand/or attenuating diseases or conditions associated with one or morecomponents of the RAS, particularly one or more angiotensin peptidemoieties. The invention further provides vaccination methods forpreventing and/or attenuating diseases or conditions in individuals,particularly in animals such as mammals, and particularly humans. In apreferred embodiment, the conjugates and conjugates of the inventionstimulate an immune response leading to the production of immunemolecules, including antibodies, that bind to one or more angiotensinpeptide moieties. The invention further provides vaccination methods forpreventing and/or attenuating diseases or conditions associated with theRAS in individuals.

The nature or type of immune response is not a limiting factor of thisdisclosure. The desired outcome of a therapeutic or prophylactic immuneresponse may vary according to the disease, according to principles wellknown in the art. Without the intention to limit the present inventionby the following mechanistic explanation, the inventive conjugates mightinduce antibodies which bind to more than one angiotensin peptidespecies thereby blocking all relevant species of angiotensin at the sametime. Alternatively the induced antibodies might bind specifically tothe C-terminus of angiotensinogen, angiotensin I or angiotensin II.Under these conditions, the induced antibodies will block activation ofangiotensinogen or angiotensin I by renin or ACE, respectively.Nevertheless, proteases different from ACE or renin such asendopeptidases and aminopeptidases can degrade angiotensinogen,angiotensin I or angiotensin II from the N-terminus thus preventing theaccumulation of antibody-bound intact angiotensinogen, angiotensin I orangiotensin II.

Furthermore, it may be desired to stimulate different types of immuneresponse depending on the disease, and according to principles known inthe art. It is well known, for example, that some immune responses aremore appropriate for a particular antigen than other immune responses.Some immune responses are, indeed, inappropriate and can causepathology, such as pathologic inflammation.

The nature of the immune response can be affected by the nature of theantigen, route of introduction into the body, dose, dosage regimen,repetitive nature of the antigen, host background, and signaling factorsof the immune system. Such knowledge is well known in the art. As such,an immune response may be tailored by the application of both art knowntheory and routine experimentation.

Furthermore, the invention embodies the use of differing core particlesduring the course of vaccination against angiotensin peptide moieties.Individuals who develop strong immune responses against core particlessuch as e.g. pili, may be immunized with conjugates comprising the sameangiotensin peptide moiety but differing in core particle.

While not wishing to be bound by theory, the current conjugates of thepresent invention provide particular novel and surprising advantages ascomponents of pharmaceutical conjugates to generate an immune response,and particularly as vaccines, against one or more angiotensin peptidemoieties. Other carriers known in the art including BSA, keyhole limpethemocyanin, tetanus toxoid, bacterial outermembrane proteins, choleratoxin, and Pseudomonas aeruginose Exotoxin A may be inappropriate foruse in an individual, and in particular a human. The aforementionedcarriers may induce allergic reactions, or stimulate pathologic immuneresponses (for example, cholera toxin, KLH, BSA). The aforementionedcarriers may require the presence of adjuvants such as complete Freund'sadjuvant, now considered inappropriate for use in humans. A number ofthe carriers may be components of current vaccines (for example, tetanustoxoid, cholera toxin, Exotoxin A). As such, an individual may possess ahigh level of pre-existing immunity to these carriers, such thatimmunization with an antigen-carrier conjugate will induce a relativelygreater immune response to the carrier than to the novel antigen. Forthese reasons, individually or as a whole, the conjugates and conjugatesof the present invention represent a useful improvement over theabove-described carrier proteins.

In the use of the embodiments of the invention, one or more angiotensinpeptide moieties conjugated to core particles can be taken up by antigenpresenting cells and thereby stimulate T-cell help to induce immuneresponses. T helper cell responses can be divided into type 1 (T_(H)1)and type 2 (T_(H)2) T helper cell responses (Romagnani, Immunol. Today18:263-266 (1997)). T_(H)1 cells secrete interferon-gamma and othercytokines which trigger B cells to produce IgG1-3 antibodies. Incontrast, a critical cytokine produced by T_(H)2 cells is IL-4, whichdrived B cells to produce IgG4 and IgE. In many experimental systems,the development of T_(H)1 and T_(H)2 responses is mutually exclusivesince T_(H)1 cells suppress the induction of T_(H)2 cells and viceversa. Thus, antigens that trigger a strong T_(H)1 responsesimultaneously suppress the development of T_(H)2 responses and hencethe production of IgE antibodies. Interestingly, virtually all virusesinduce a T_(H)1 response in the host and fail to trigger the productionof IgE antibodies (Coutelier et al., J. Exp. Med. 165:64-69 (1987)).Antibodies of the IgE isotype are important components in allergicreactions. Mast cells bind IgE antibodies on their surface and releasehistamines and other mediators of allergic response upon binding ofspecific antigen to the IgE molecules bound on the mast cell surface.The isotype pattern typical of T_(H)1 responses is not restricted tolive viruses but has also been observed for inactivated or recombinantviral particles (Lo-Man et al., Eur. J. Immunol. 28:1401-1407 (1998)).Thus, by using the processes of the invention (e.g., AlphaVaccineTechnology), viral particles can be decorated with various angiotensinpeptide moieties and used for immunization. Due to the resulting “viralstructure” of the array, a T_(H)1 response will be elicited,“protective” IgG1-3 antibodies will be produced, and the production ofIgE antibodies which cause allergic reactions will be prevented. Thus,the invention embodies conjugates capable of inducing preferred immuneresponses, notably T_(H)1 type responses. Futher, the invention embodiesthe use of conjugates of the invention to counter allergic reactionsinduced by alternative vaccines against antigens of interest.

A further advantageous feature of the invention is that angiotensinpeptide moieties may be presented on the particles in regular,repetitive arrays that are able to induce efficient immune responsesboth with and without T-cell help. This feature of the invention isparticularly advantageous.

Unlike isolated proteins, viruses induce prompt and efficient immuneresponses in the absence of any adjuvants both with and without T-cellhelp (Bachmann & Zinkernagel, Ann. Rev. Immunol: 15:235-270 (1997)).Although viruses often consist of few proteins, they are able to triggermuch stronger immune responses than their isolated components. ForB-cell responses, it is known that one crucial factor for theimmunogenicity of viruses is the repetitiveness and order of surfaceepitopes. Many viruses exhibit a quasi-crystalline surface that displaysa regular array of epitopes which efficiently crosslinksepitope-specific immunoglobulins on B cells (Bachmann & Zinkernagel,Immunol. Today 17:553-558 (1996)). This crosslinking of surfaceimmunoglobulins on B cells is a strong activation signal that directlyinduces cell-cycle progression and the production of IgM antibodies.Further, such triggered B cells are able to activate T helper cells,which in turn induce a switch from IgM to IgG antibody production in Bcells and the generation of long-lived B cell memory—the goal of anyvaccination (Bachmann & Zinkernagel, Ann. Rev. Immunol. 15:235-270(1997)). The present invention provides one way to improve theefficiency of vaccination by increasing the degree of repetitiveness ofthe antiogensin peptide moiety to be used for immunization, throughbinding of the angiotensin peptide moiety to the core particles. Aspreviously noted, the invention provides for conjugates comprising coreparticle modified to alter the number and or arrangement of theorganizer.

As would be understood by one of ordinary skill in the art, whenconjugates of the invention are administered to an individual, they maybe in a conjugate which contains salts, buffers, adjuvants, or othersubstances which are desirable for improving the efficacy of theconjugate. Examples of materials suitable for use in preparingpharmaceutical conjugates are provided in numerous sources including,REMINGTON'S PHARMACEUTICAL SCIENCES (Osol, A, ed., Mack Publishing Co.,(1990)).

Conjugates of the invention are said to be “pharmacologicallyaccept-able” if their administration can be tolerated by a recipientindividual. Further, the conjugates of the invention will beadministered in a “therapeutically effective amount” (i.e., an amountthat produces a desired physiological effect).

To induce an immune response, the conjugates of the present inventionmay be administered to an animal, suitably a mammal such as a human, byvarious methods known in the art, but will normally be administered byinjection, infusion, inhalation, oral administration, or other suitablephysical methods. The conjugates may alternatively be administeredintramuscularly, intravenously, transmucosally, transdermally orsubcutaneously. Components of conjugates for administration includesterile aqueous (e.g., physiological saline) or non-aqueous solutionsand suspensions. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Carriers or occlusive dressings canbe used to increase skin permeability and enhance antigen absorption.

Further embodiments of the invention include immune molecules producedby immunization with conjugates of the invention. Immune moleculesinclude antibodies and T-cell receptors. Such immune molecules may beuseful in a vaccinated individual for binding to target one or moreangiotensin peptide moieties. Immune molecules may also be useful whentransferred to another individual not immunized against conjugates orconjugates of the invention, thereby to “passively” transfer immunity.In one embodiment, the immune molecule is an antibody. A monoclonalantibody suitable for binding one or more angiotensin peptide moietiesmay be transferred into an individual to achieve therapy or prophylaxis.

The invention also encompasses the use of antibodies produced byimmunization with the conjugates or conjugates of the invention in kitsfor the detection of one or more angiotensin peptide moieties inimmunoassays (eg ELISA). In a related embodiment, repetitive orderedarrays of angiotensin peptide moieties can be useful for the detectionof antibodies against such moieties in binding assays. Other embodimentsof the invention include processes for the production of the conjugatesof the invention and methods of medical treatment using such conjugates,particularly to treat one or more physical disorders associated with theRAS such as hypertension, stroke, infarction, congestive heart failure,kidney failure or retinal hemorrhage.

It will be understood by one of ordinary skill in the relevant arts thatother suitable modifications and adaptations to the methods andapplications described herein are readily apparent and may be madewithout departing from the scope of the invention or any embodimentthereof. Having now described the present invention in detail, the samewill be more clearly understood by reference to the following examples,which are included herewith for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES Example 1 Coupling of Peptides Derived from Angiotensin I andAngiotensin II to Qβ and the Immunization of Mice with the ResultingConjugates

A. Production of Conjugates

The following angiotensin peptides moieties were chemically synthesized:

CGGDRVYIHPF (“Angio 1”), CGGDRVYIHPFHL (“Angio 2”), DRVYIHPFHLGGC(“Angio 3”), CDRVYIHPFHL (“Angio 4”), CHPFHL (“Angio 5”), CGPFHL (“Angio6”), CYIHPF (“Angio 7”), CGIHPF (“Angio 8”), CGGHPF (“Angio 9”),DRVYIGGC (“Angio 13”), DRVYGGC (“Angio 14”) and DRVGGC (“Angio 15”).They were used for chemical coupling to Qβ as described in thefollowing.

For peptides Angio 1 to Angio 4: A solution of 5 ml of 2 mg/ml Qβ capsidprotein in 20 mM Hepes. 150 mM NaCl pH 7.4 was reacted for 30 minuteswith 507 μl of a solution of 13 mg/ml Sulfo-MBS (Pierce) in H₂O at 25°C. on a rocking shaker. The reaction solution was subsequently dialyzedtwice for 2 hours against 2 L of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4°C. 665 μl of the dialyzed reaction mixture was then reacted with 2.8 μlof each of the corresponding 100 mM peptide stock solution (in DMSO) fortwo hours at 25° C. on a rocking shaker. The reaction mixture wassubsequently dialyzed 2×2 hours against 2 liters of 20 mM Hepes, 150 mMNaCl, pH 7.4 at 4° C.

For peptides Angio 5-9 and Angio 13-15: A solution of 3 ml of 2 mg/ml Qβcapsid protein in 20 mM Hepes. 150 mM NaCl pH 7.2 was reacted for 50minutes with 86 μl of a solution of 100 mM SMPH(succinimidyl-6-(β-maleimidopropionoamido hexanoate, Pierce) in DMSO at25° C. on a rocking shaker. The reaction solution was subsequentlydialyzed twice for 2 hours against 2 L of 20 mM Hepes, 150 mM NaCl, pH7.2 at 4° C. 514 μl of the dialyzed reaction mixture was then reactedwith 3.6 μl of each of the corresponding 100 mM peptide stock solution(in DMSO) for 4 hours at 25° C. on a rocking shaker. The reactionmixture was subsequently dialyzed 2×2 hours against 2 liters of 20 mMHepes, 150 mM NaCl, pH 7.2 at 4° C.

B. Immunization

Female Balb/c mice were vaccinated with one of the nine angiotensinpeptide derivatives coupled to Qβ capsid protein without the addition ofadjuvants. 50 g (Qβ-Angio 1-4 vaccine) or 20 μg (Qβ-Angio 5-9 vaccine)of total protein of each sample was diluted in PBS to 200 μl andinjected subcutaneously (100 μl on two ventral sides) on day 0 and day14. Mice were bled retroorbitally on day 21 and their serum was analyzedusing an antgiotensin-specific ELISA

It should be noted that the human and the murine sequences of theangiotensin peptides identically correspond to each other. Therefore,immunization of a human or a mouse with vaccines or conjugates,respectively, comprising angiotensin peptide moieties as antigenicdeterminant in accordance with the invention, is a vaccination against aself-antigen.

Example 2 ELISA Analysis of Sera from Mice Vaccinated with PeptidesDerived from Angiotensin I and Angiotensin II Coupled to Qβ

Angio 1 to Angio 9 and Angio13-15 peptide derivatives prepared asdescribed in Example 1 were individually coupled to bovine RNAse A(Sigma) using the chemical cross-linker sulfo-SPDP. ELISA plates werecoated overnight at 4° C. with coupled RNAse preparations at aconcentration of 10 μg/ml in coating buffer (0.1M NaH₂CO₃, pH 9.6).Alternatively, angiotensin I or angiotensin II (SIGMA) were diluted inthe same coating buffer to a concentration of 200 μg/ml. The plates wereblocked with blocking buffer (2% bovine serum albumin (BSA) in PBS (pH7.4)/0.05% Tween 20) for 2 hours at 37° C., washed with PBS (pH7.4)/0.05% Tween 20 and then incubated for 2 hours at room temperaturewith serially diluted mouse sera in blocking buffer. The plates werewashed with PBS (pH 7.4)/0.05% Tween 20. and then incubated with horseradish peroxidase-labeled goat anti-mouse IgG antibody at 1 μg/ml(Jackson ImmunoResearch) for 1 hour at room temperature. The plates werewashed with PBS (pH 7.4)/0.05% Tween 20 and the substrate solution wasadded (0.066M Na₂HPO₄, 0.035 M citric acid (pH 5.0)+0.4 mg OPD(1,2-Phenylenediamine dihydrochloride)+0.01% H₂O₂). After 10 min thecolor reaction was stopped with 5% H₂SO₄ and absorbance was read at 450nm.

As a control, preimmune sera of the same mice were also tested. ControlELISA experiments using sera from mice immunized with unrelated peptidescrosslinked to Qβ or other carriers showed that the antibodies detectedwere specific for the respective peptide. ELISA titers were calculatedas the reciprocal serum dilution which gives a half-maximal signal inthe ELISA (50% of maximal optical density).

Results:

FIG. 1 shows ELISA analyses of IgG antibodies specific for the Angio 2peptide and angiotensin I in sera of mice immunized with Angio 1-4peptides coupled to Qβ capsid protein. Qβ-Angio 1, Qβ-Angio 1, Qβ-Angio3 and Qβ-Angio 4, as used in the figures, stand for the vaccine injectedin the mice, from which the sera are derived in accordance with abovedefinition of the angiotensin peptides. Female Balb/c mice werevaccinated subcutaneously with 50 μg of vaccine in PBS on day 0 and day14. IgG antibodies in sera of mice vaccinated with Qβ-Angio 1, Qβ-Angio2, Qβ-Angio 3 and Qβ-Angio 4 were measured on day 21 against Angio 2peptide coupled to RNAse A and against angiotensin I. As a control, apre-immune sera were also analyzed. Results for indicated serumdilutions are shown as optical density at 450 nm. The average of threemice each (including standard deviations) is shown for Angio 2. Theaverage of two mice each are shown for angiotensin I. All vaccinatedmice made specific IgG antibodies against the Angio 2 peptide as well asangiotensin I although the mice immunized with the Angio 2, Angio 3 orAngio 4 peptide exhibited higher titers than those vaccinated with theAngio 1 peptide correlating with the close similarity of the Angio 2,Angio 3 and Angio 4 peptides and angiotensin I.

FIG. 2 shows ELISA analyses of IgG antibodies specific for the Angio Ipeptide and angiotensin II in sera of mice immunized with Angio 1-4peptides coupled to Qβ capsid protein. Qβ-Angio 1, Qβ-Angio 1, Qβ-Angio3 and Qβ-Angio 4, as used in the figures, stand for the vaccine injectedin the mice, from which the sera are derived in accordance with abovedefinition of the angiotensin peptides. Female Balb/c mice werevaccinated subcutaneously with 50 μg of vaccine in PBS on day 0 and day14. IgG antibodies in sera of mice vaccinated with Qβ-Angio 1, Qβ-Angio2, Qβ-Angio 3 and Qβ-Angio 4 were measured on day 21 against Angio 1peptide coupled to RNAse A and against angiotensin II. As a control, apre-immune sera were also analyzed. Results for indicated serumdilutions are shown as optical density at 450 nm. The average of threemice each (including standard deviations) is shown for Angio 1. Theaverage of two mice each are shown for angiotensin II. All vaccinatedmice made specific IgG antibodies against the Angio 1 peptide as well asangiotensin II although the mice immunized with the Angio 1 peptideexhibited the highest titers correlating with the close similarity ofthe Angio 1 peptide and angiotensin II.

FIG. 3 shows ELISA analyses of IgG antibodies specific for the Angio 2peptide and angiotensin I in sera of mice immunized with Angio 5-9peptides coupled to Qβ capsid protein. Qβ-Angio 5, Qβ-Angio 6, Qβ-Angio7, Qβ-Angio 8 and Qβ-Angio 9, as used in the figures, stand for thevaccine injected in the mice, from which the sera are derived inaccordance with above definition of the angiotensin peptides. FemaleBalb/c mice were vaccinated subcutaneously with 20 μg of vaccine in PBSon day 0 and day 14. IgG antibodies in sera of mice vaccinated withQβ-Angio 4, Qβ-Angio 5, Qβ-Angio 6, Qβ-Angio 7, Qβ-Angio 8 and Qβ-Angio9 were measured on day 21 against Angio 2 peptide coupled to RNAse A andagainst angiotensin I. Results for indicated serum dilutions are shownas optical density at 450 nm. The average of two mice each are shown.The two mice vaccinated with Qβ-Angio 8 and Qβ-Angio 9 exhibited verylow or no specific titers against the Angio 2 peptide as well asangiotensin I, indicating that these two types of vaccine inducedantibodies which were mostly specific for the C-terminus of angiotensinII but not for angiotensin I (see also FIG. 4).

FIG. 4 shows ELISA analyses of IgG antibodies specific for the Angio 1peptide and angiotensin II in sera of mice immunized with Angio 5-9peptides coupled to Qβ capsid protein. Qβ-Angio 5, Qβ-Angio 6, Qβ-Angio7, Qβ-Angio 8 and Qβ-Angio 9, as used in the figures, stand for thevaccine injected in the mice, from which the sera are derived inaccordance with above definition of the angiotensin peptides. FemaleBalb/c mice were vaccinated subcutaneously with 20 μg of vaccine in PBSon day 0 and day 14. IgG antibodies in sera of mice vaccinated withQβ-Angio 4, Qβ-Angio 5, Qβ-Angio 6, Qβ-Angio 7, Qβ-Angio 8 and Qβ-Angio9 were measured on day 21 against Angio 1 peptide coupled to RNAse A andagainst angiotensin II. Results for indicated serum dilutions are shownas optical density at 450 nm. The average of two mice each are shown.The two mice vaccinated with Qβ-Angio 5 and Qβ-Angio 6 exhibited verylow or no specific titers against the Angio I peptide as well asangiotensin II, indicating that these two types of vaccine inducedantibodies which were mostly specific for the C-terminus of angiotensinI but not for angiotensin II (see also FIG. 3).

The following table summarizes the ELISA analysis of sera from micevaccinated with the Angio peptides 1 to 9 coupled to Qβ. Average ELISAtiters from day 21 were calculated as described in Example 2. TABLE 1Angiotensin-derived peptides used for vaccination of mice and resultingantibody responses against the used peptides as well as angiotensin Iand angiotensin II. Avg. ELISA Avg. ELISA Avg. ELISA Avg. ELISA Name ofAmino acid titer against titer against titer against titer againstpeptide sequence Angiotensin I Angio 2 peptide Angiotensin II Angio 1peptide Angio 1 CGGDRVYIHPF <50 5321 711 13975 Angio 2 CGGDRVYIHPFHL1250 16416 68 1064 Angio 3 DRVYIHPFHLGGC 1250 20898 74 476 Angio 4CDRVYIHPFHL 559 11898 142 906 Angio 5 CHPFHL 3856 1877 50 <50 Angio 6CGPFHL 1250 870 <50 <50 Angio 7 CYIHPF 112 626 6250 971 Angio 8 CGIHPF<50 87 476 1350 Angio 9 CGGHPF <50 50 476 2338 Angio 13 DRVYIGGC  n.t.*n.t. n.t. n.t. Angio 14 DRVYGGC n.t. n.t. n.t. n.t. Angio 15 DRVGGC n.t.n.t. n.t. n.t.*n.t. = not tested

The results with Angio 5 and Angio 6 show that peptides can be inducedthat selectively recognize Angiotensin I. Furthermore, the results withAngio 7-9 show that antibodies can be induced that selectively recognizeAngiotensin II but not Angiotensin I. Since Angiotensin I and II differby 2 amino acids only at the C-terminus while the remaining 8 aminoacids are identical, these results demonstrate that all antibodiesinduced by Angio 5 or Angio 6 selectively recognize the C-terminus ofAngiotensin I and that antibodies induced by Angio 7-9, and inparticular Angio 8-9, selectively recognize the C-terminus ofAngiotensin II. Thus, the shared 8 amino acids are not recognized and inparticular the shared N-terminus is not recognized. This indicates thatthe N-terminus is not buried inside antibodies when bound and,therefore, is accessible for proteases.

Having now fully described the present invention in some detail by wayof illustration and example for purposes of clarity of understanding, itwill be obvious to one of ordinary skill in the art that the same can beperformed by modifying or changing the invention within a wide andequivalent range of conditions, formulations and other parameterswithout affecting the scope of the invention or any specific embodimentthereof, and that such modifications or changes are intended to beencompassed within the scope of the appended claims.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1. An angiotensin peptide moiety-carrier conjugate comprising: (a) acarrier with at least one first attachment site, and (b) at least oneangiotensin peptide moiety with at least one second attachment site;wherein said carrier comprises a core particle that is a virus-likeparticle of an RNA bacteriophage and; wherein said second attachmentsite associates with said first attachment site through at least onenon-peptide bond so as to form an ordered and repetitive angiotensinpeptide moiety-carrier conjugate. 2-7. (canceled)
 8. The conjugate ofclaim 1, wherein said virus-like particle of an RNA bacteriophagecomprises proteins, or fragments thereof, of an RNA bacteriophage. 9.The conjugate of claim 8, wherein said RNA bacteriophage is selectedfrom the group consisting of: (a) bacteriophage Qβ; (b) bacteriophageR17; (c) bacteriophage fr; (d) bacteriophage GA; (e) bacteriophage SP;(f) bacteriophage MS2; (g) bacteriophage M11; (h) bacteriophage MX1; (i)bacteriophage NL95; (O) bacteriophage f2; (k) bacteriophage AP205; and(l) bacteriophage PP7.
 10. The conjugate of claim 1, wherein saidvirus-like particle of an RNA bacteriophage comprises recombinantproteins, or fragments thereof, of bacteriophage Qβ.
 11. The conjugateof claim 1, wherein said virus-like particle of an RNA bacteriophagecomprises recombinant proteins or fragments thereof, of bacteriophageAP205.
 12. The conjugate of claim 8, wherein said virus-like particle ofan RNA bacteriophage comprises one or more coat proteins of said RNAbacteriophage which have been modified by deletion or substitution toremove at least one naturally occurring lysine residue or that have beenmodified by insertion or substitution to add at least one lysineresidue.
 13. (canceled)
 14. (canceled)
 15. The conjugate of claim 10,wherein said recombinant proteins comprise one or more coat proteinshaving an amino acid sequence of SEQ ID NO:3.
 16. The conjugate of claim10, wherein said recombinant proteins comprise a mixture of coatproteins having amino acid sequences of SEQ ID NO: 4 or mutants thereof,and SEQ ID NO:3.
 17. The conjugate of claim 12 wherein said RNAbacteriophage is Qβ.
 18. (canceled)
 19. (canceled)
 20. The conjugate ofclaim 17, wherein said virus-like particle comprises one or moreproteins having an amino acid sequence selected from the groupconsisting of: (a) the amino acid sequence of SEQ ID NO:6; (b) the aminoacid sequence of SEQ ID NO:7; (c) the amino acid sequence of SEQ IDNO:8; (d) the amino acid sequence of SEQ ID NO:9; and (e) the amino acidsequence of SEQ ID NO:10.
 21. (canceled)
 22. The conjugate of claim 1,wherein said first attachment site comprises: (a) an amino group; (b) acarboxyl group; (c) a sulfhydryl group; (d) a hydroxy group; (e) aguanidinyl group; or (f) a histidinyl group.
 23. The conjugate of claim1, wherein said at least one first attachment site is selected from thegroup consisting of a lysine residue, an arginine residue, a cysteineresidue, an asparatate residue, a glutamate residue, a serine residue, athreonine residue, a histidine residue and a tyrosine residue.
 24. Theconjugate of claim 1, wherein said at least one first attachment site isa lysine residue. 25-33. (canceled)
 34. The conjugate of claim 1,wherein said angiotensin peptide moiety is an angiotensin peptideselected from the group consisting of angiotensinogen, angiotensin I,angiotensin II, and fragments or derivatives thereof.
 35. The conjugateof claim 1, wherein said angiotensin peptide moiety with said secondattachment site has an amino acid sequence selected from the groupconsisting of: (a) CGGDRVYIHPF; (SEQ ID NO: 19) (b) CGGDRVYHPFHL; (SEQID NO: 20) (c) DRVYIHPFHLGGC; (SEQ ID NO: 21) (d) CDRVYIHPFHL; (SEQ IDNO: 22) (e) CHPFHL; (SEQ ID NO: 23) (f) CGPFHL; (SEQ ID NO: 24) (g)CYIHPF; (SEQ ID NO: 25) (h) CGIHPF; (SEQ ID NO: 26) (i) CGGHPF; (SEQ IDNO: 27) (j) DRVYIGGC; (SEQ ID NO: 28) (k) DRVYGGC; (SEQ ID NO: 29) (l)DRVGGC; (SEQ ID NO: 30) (m) DRVY; (SEQ ID NO: 34) (n) DRVYIHPF; (SEQ IDNO: 17) and (o) DRVYIHPFHL. (SEQ ID NO: 16)


36. A pharmaceutical composition comprising one or more of theconjugates of claim 1 and a pharmaceutically acceptable carrier orexcipient.
 37. A vaccine composition comprising the conjugate of claim 1and an immunologically acceptable carrier or excipient.
 38. The vaccinecomposition of claim 37, wherein said vaccine composition furthercomprises at least one adjuvant.
 39. A method of immunizing an animalagainst an angiotensin peptide moiety, comprising administering theconjugate of claim 1 to an animal under conditions such that said animaldevelops an immune response to said angiotensin peptide moiety.
 40. Themethod of claim 39, wherein said conjugate is administered to saidanimal via a route of administration selected from the group consistingof intranasal administration, oral administration, subcutaneousadministration, transdermal administration, intramuscular administrationand intravenous administration.
 41. The method of claim 40, wherein saidconjugate is administered to said animal intranasally.
 42. A method ofimmunizing an animal against an angiotensin peptide moiety, comprisingadministering the vaccine composition of claim 37 or claim 38 to ananimal under conditions such that said animal develops an immuneresponse to said angiotensin peptide moiety.
 43. The method of claim 42,wherein said composition is administered to said animal via a route ofadministration selected from the group consisting of intranasaladministration, oral administration, subcutaneous administration,transdermal administration, intramuscular administration and intravenousadministration.
 44. The method of claim 42, wherein said composition isadministered to said animal intranasally.
 45. A method of treating orpreventing a physical disorder associated with the renin-activatedangiotensin system comprising administering to an animal atherapeutically or prophylactically effective amount of one or more ofthe conjugates of claim
 1. 46. The method of claim 45, wherein saidphysical disorder associated with the renin-activated angiotensin systemis selected from the group consisting of hypertension, stroke,infarction, congestive heart failure, kidney failure and retinalhemorrhage.
 47. A method of treating or preventing a physical disorderassociated with the renin-activated angiotensin system comprisingadministering to an animal a therapeutically or prophylacticallyeffective amount of the pharmaceutical composition of claim
 36. 48. Amethod of treating or preventing a physical disorder associated with therenin-activated angiotensin system comprising administering to an animalan immunologically effective amount of the vaccine composition of claim37 or claim
 38. 49. The method of claim 47 wherein said physicaldisorder associated with the renin-activated angiotensin system isselected from the group consisting of hypertension, stroke, infarction,congestive heart failure, kidney failure and retinal hemorrhage.
 50. Theconjugate of claim 1, wherein said second attachment site associateswith said first attachment site through at least one non-peptidecovalent bond so as to form an ordered and repetitive angiotensinpeptide moiety-carrier array.
 51. The conjugate of claim 1, wherein saidfirst attachment comprises an amino group.
 52. The conjugate of claim 1,wherein said second attachment site comprises a sulfhydryl group. 53.The conjugate of claim 1, wherein said first attachment site comprisesan amino group and wherein said second attachment comprises a sulfhydrylgroup.
 54. The conjugate of claim 1, wherein said first attachment siteis not a sulfhydryl group.
 55. The conjugate of claim 1, wherein saidvirus-like particle of an RNA bacteriophage comprises recombinantproteins, or fragments thereof, of bacteriophage fr.
 56. The conjugateof claim 1, wherein only one of said second attachment sites associateswith said first attachment site through at least one non-peptidecovalent bond leading to a single and uniform type of binding of saidangiotensin peptide moiety to said core particle, wherein said only onesecond attachment site that associates with said first attachment siteis a sulfhydryl group, and wherein said angiotensin peptide moiety andsaid core particle interact through said association to form an orderedand repetitive antigen array.
 57. The conjugate of claim 1, wherein saidangiotensin peptide moiety is angiotensin II.
 58. The conjugate of claim1, wherein said angiotensin peptide moiety with said second attachmentsite consists of the amino acid sequence of CGGDRVYIHPF (SEQ ID NO: 19).59. The vaccine composition of claim 38, wherein said adjuvant isaluminium hydroxide.
 60. The method of claim 48 wherein said physicaldisorder associated with the renin-activated angiotensin system isselected from the group consisting of hypertension, stroke, infarction,congestive heart failure, kidney failure and retinal hemorrhage.